Retrospective Theses and Dissertations Iowa State University Capstones, Theses andDissertations

1-1-1973

Venezuelan equine encephalomyelitis : the goat as asentinel for virus activity and as a serum donor forfluorescent antibody conjugatesGene Allan EricksonIowa State University

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Venezuelan equine encephalomyelitis:

The goat as a sentinel for virus

activity and as a serum donor for

fluorescent antibody conjugates

by

Gene Allan Erickson

A Thesis Submitted to the

Graduate Faculty in Partial Fulfi 1 lment of

The Requirements for the Degree of

MASTER OF SCIENCE

Department: Veterinary Microbiology and Preventive Medicine

Major: Veterinary Microbiology

Signatures have been redacted for privacy

lewa State University Ames, Iowa

1973

I I

.ISl-l 1'173 £..,. 44 G,b INTRODUCTION

REVIEW OF LITERATURE

MATERIALS AND METHODS

RESULTS

DISCUSS I ON

SUMMARY

APPENDIX

B I B LI OGRAPHY

ACKNOWLEDGEMENTS

i i

TABLE OF CONTENTS

Page

3

28

43

68

74

76

89

101

i i i

TABLE OF CONTENTS

Table 1. Antigenic subtypes of Venezuelan equine encephalomyelitis virus

Table 2. Mosquito species from which epizoodemic VEE viruses have been isolated

Table 3. Mosquito infection and transmission rates for VEE virus IA

Page

4

8

10

Table 4. Mosquito infection thresholds for VEE virus IB 10

Table 5. Mammals considered to be involved in the 21 maintenance of endemic VEE virus activity

Table 6. Mosquito species from which endemic VEE virus have been isolated

23

Table 7. Susceptibility of different cell types to 44 GJ9-1BJ VEE virus

Table 8. Virus·titers in serum of goats inoculated with 53 GJ9~1BJ VEE virus

Table 9. Body temperature of goat number 159 inoculated 53 with IB VEE virus

Table 10. Virus detection by the fluorescent antibody test 67 (FAT) and by intracerebral inoculation of suck! ing mice

Figure 1.

Figure 2.

Figure 3.

Figure 4.

iv

LIST OF FIGURES

Sentinel goat study. TC-83 SN antibody titers of goats 147, 152, 157, 158, 159, and 161 between days 6 and 45 postinoculation. Titers 1 i sted were the highest serum <! i 1 ut ion that produced at least 90% plaque reduction. See Appendix for specific titers.

Sentinel goat study. GJ9-1BJ SN antibody titers of goats 147, 152, 157, 158, 159, and 161 between days 6 and 45 postinoculation. Titers listed were the highest serum dilution that produced at least 90"/o plaque reduction. See Appendix for speci~ic titers.

Sentinel goat stud)'.. Hi antibody titers of goats 147, 152, 157, 158, 159 and 161 between days 6 and 45 post inoculation. See Appendix for specific titers.

Absence of clinical signs in a domestic goat three days after infection with the GJ9-1BJ strain of Venezuelan equine encephalomyelitis (VEE) virus.

Figure 5. VEE conjugate applied to normal BHK-21 cell monolayer. Note absence of specific fluorescence. 125x

Page

47

49

52

56

56

Figure 6. VEE conjugate applied to GJ9-1BJ VEE-virus- 58 i.nfected BHK-21 ce 11 mono 1 ayer at 24 hours postinoculation. Note specific perlnuclear and cytoplasmic fluorescence. 125x

Figure 7. Normal conjugate applied to GJ9-1BJ VEE-virus- 58 infected BHK-21 cell monolayer at 24 hours postinoculation. Note absence of specific fluorescence. 125x

Figure 8. VEE conjugate applied to TC-83-VEE-virus- 60 infected BHK-21 cell monolayer at 24 hours postinoculation. Note specific, granular perinuclear and cytoplasmic fluorescence. 500x

v

Figure 9. VEE conjugate applied to GJ9-1BJ-,VEE-virus-infected BHK-21 cell monolayer at 24 hours postinoculation. Note specific, granular perinuclear fluorescence. 1188x

Figure 10. VEE conjugate applied to TC-83-VEE-virus-infected BHK-21 cell monolayer at 48 hours' postinoculation. Note specific cytoplasmic fluorescence of rounded up cells. 125x

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60

62

Figure 11. VEE conjugate applied to TC-83-VEE-virus- 62 infected BHK-21 cell monolayer at 48 hours postinoculation. Note specific, granular cytoplasmic fluorescence of rounded up cells. SOOx

Figure 12. VEE conjugate applied to western equine en-cephalomyelitis virus-infected BHK-21 cell, monolayer at 24 hours postlnoculatlon. Note absence of cross-staining. 125x

Figure 13. VEE conjugate applied to eastern equine en-cephalomyelitis virus-infected BHK-21 cell monolayer at 24 hours postinoculation. Note absence of cross-staining. 125x

64

64

INTRODUCTION

The insidious spread of Venezuelan equine encephalomyelitis virus

(VEE) from South America through Central America to the.United States has

aroused great concern within the horse industry and among agricultural

officials in the United States. Indeed the disease in the epizoodemic

form appears to have been eliminated from the continental borders of this

country but the mechanisms by which this virus can apparently reside

within a given region are unknown. The term epizoodemic as employed in

the text above refers to the concurrent presence of an epidemic and an

epizootic·due to a single disease agent (56,57). There is at present a

great confusion of terminology in the literature due to the remarkable

ability of the VEE virus complex to give rise to several forms of disease

among man, domestic animals, and fauna of the countryside. Therefore, in

an attempt to reduce this confusion, the term epizoodemic shall be em-

ployed in this study.

Due to the explosiveness of epizoodemic VEE it has become imperative

to have rapid diagnostic.techniques available to the virologist for con-

firmation of the disease agent in domestic livestock and in wildlife

populations. Such a technique is readily available in the form of the

fluorescent antibody test whf.ch can be used to obtain positive or negative.

results within a 48 hour period as compared to suckling mouse inoculation.

with complement fixation (CF) test confirmation which requires from 72

hours to one week for comp 1 et ion of diagnosis •.

-'

2

The invasion of the United States by VEE with subsequent eradication

in 1971 has emphasized the need for a natural sentinel to detect the

virus should it reenter the country in the future. Several animals have

been investl'gated as possible sentinels by other workers. These include

the canine (9), bovine, porcine (33), equine (25), laboratory (39,43,105,

107,108) and wild ungulate (67) species. Despite such intensive work, the

suitability of the goat, (Capra hircus), as a sentinel species has not

been investigated.

In hypothesizing the means by which epizoodemic strains of VEE run

rampant through the countryside, reference has occasionally been made to

the possibility of the goat acting as ,a silent amplifier of the virus

(57,104,110). ·Jt was the purpose of this. study, firstly, to investigate

the suitability of the goat as a sentinel for virus activity, and

secondly, to determine the sensitivity of the fluorescent antibody test

on cell cultures as compared· to suckling mouse inoculation with con-

firmatory CF test for identification of VEE virus from tissue specimens.

3

REVIEW OF LITERATURE

History

An encephalitic disease of equines which appeared in 1935 in the

river valleys of Huila, Tolima, Valle, and Bolivar in the Andes mountains

of western Colombia was tentative I y diagnosed as Barna' s disease or

European equine encephalitis. From there the disease spread to Magdalena,

Colombia in 1936 and later that year appeared in the Guajira peninsula of

Colombia and Venezuela ( 125). In 1938 Kubes. and Rios of the Venezuelan

Ministry of Agriculture and Animal Husbandry isolated a filterable v.irus

from the brain of a horse that had died with encephalitic signs (77).

Subsequent characterization of the agent was done by Kubes (76),

Kubes and R las ( 77) and by Beck and Wyckoff ( 6) in 1939 and 1944. The

virus was found to be of greater virulence for equidae than any of the

eastern or western equine encephalomyelitis viru.ses previously ·identified·

in South America. It also differed immunologically from these viruses.

Due to its origin the encephalitic agent was designated "Venezuelan equine

encephalomyel it is virus. 11 A uniform system of nomenclature was not

available for the complex of endemic (enzootic) and epizoodemic strains

of Venezuelan equine encephalomyelitis (VEE) virus that later arose

until Youn,g and Johnson (134) in 1969 published their system embracing

the antigenic variants of VEE (Table 1).

Since 1939, either epizoodemics or epidemics of VEE have been

described in Peru, Ecuador, Columbia, Venezuela, Trinidad, Costa Rica,

Nicaragua, Honduras, El Salvador, Guatemala, Mexico and the United States.

4

Table 1. Anti gen i c· subtypes of Venezuelan ·equine enceph a 1omye1 it is virus (69, 134)

Antigenic Isolate Origin Epizoodemic subtype

IA Trinidad Tr.inidad, 1943 Yes donkey

IB lea Peru, 1946 Yes Pergamlno Argentina, 1958 Yes

re p676 Venezuela, 1963 Yes V198 . Colombia, 1962 Yes

ID 3880 Eastern 'No Panama, 1961

IE Mena II Western No Panama, 1962

63A216 Mexico, 1963 No

II Fe3-7c Florida, 1963 No

III Mucambo Brazil, 1954 No Paramaribo Surinam, 1963 No 52049 Trinidad, 1959 No

IV Pixuna Brazil, 1961 No

5

Endemic foci have also been found to be widely distributed in the western

hemisphere including Brazil, Trinidad, Surinam, Colombia, Panama,

Nicaragua, Honduras, Guatemala, British Honduras, Mexico and Florida (57).

Current virus activity

In 1969 a severe epizoodemic of VEE arose in Guatemala and eventually

reached Texas in late June, 1971 (22,24,_37,39,42,56,75,114,124,13P). The

source of VEE virus for this and other epizoodemics remains an enigma.

Three possible alternatives for the introduction of the virulent IB

subtype of VEE virus into Guatemala have recently been proposed by

Franck and Johnson (42). They are (1) mutation of an endemic virus

subtype, (2) sudden emergence of a previously silent unrecognized virus,

and (3) introduction of the virus from another region. Introduction of

the IB subtype of VEE virus into Guatemala from Ecuador has been suggested

by several authors (37,42,86,114, 1~9).

Equine deaths were first reported along the Pacific coast of

Guatemala near El Salvador and shortly thereafter in northeastern

El Salvador. VEE then spread north and south from each focus. The

southward spread of the disease included Honduras (1969), Nicaragua

(1969), and Costa Rica (1970). Susceptible equines were vaccinated with

the attenuated TC-83 strain of VEE virus in Guatemala in an attempt to

halt the northward spread of epizoodemic VEE. However, in August, 1969,

epizoodemic VEE virus was isolated in the upper Grijalva River Valley

of Chiapas, Mexico near the Guatemalan border. Equine deaths continued

through the month of February and increased with the start of the rainy

6

season in June. From there the disease spread westward through the

mountain canyons to the state,of Oaxaca on the Pacific coast and was

confined to that region by vaccination until September when the disease

appeared just outside th'e vaccination zone in the state of Veracruz.

Vaccination was resumed only to have epizoodemic VEE occur again just

outside the barrier to the north.

Extension of the disease followed a similar pattern on the Pacific

coast as it progressively appeared beyond each new vaccination barrier.

Epizoodemic VEE had spread northward along the Pacific coast through the

state of Guerrero to the state of Michoacah by November, 1970.

Virus activity continued unreported through the end of 1970 and

into Apri 1 of 1971. Equine deaths were then confirmed· as VEE in Veracruz

and a new vaccination barrier was established to the no'rth. Despite al 1

vaccination barriers epizoodemic VEE continued its northward spread across

salt water lagoons of Mexico's gulf coast some 400 kilometers to Texas

( 125) •

First case of epizoodemic VEE in the United States

The first confirmed case of epizoodemic VEE .in the United States was

reported on June 23, 1971, near Three Rivers, Five Oak County, Texas,

approximately 320 kilometers northwest of Brownsville. The Texas

epizoodemic peaked in the equine population by mid-July (30 virus

isolations per day) and by the end of July had decreased to fewer than

5 viral isolates of epizoodemic VEE virus per day. A combination of

ultra-low-volume aerial insecticide application and vaccination (TC-83

7

virus) of all susceptible equines in Texas effectively brought the in-.

vasion of Texas' equine and human populations to an end by November 7,

1971 (75). A total of 1,620 equine deaths and an additional 2,000

clinical cases of epizoodemic VEE were recorded in Texas in 1971 (86).

Epizoodemic virus activity in Mexico in 1972

Confirmed (virus isolation) epizoodemic VEE virus activity recurred

in Mexico in the states of Durango and Sonora in 1972. Other noncon-

finned reports of VEE outbreaks came from the states of Nayarit, Guerrero,

and Morelos. Intensification of vaccination of susceptible equines with

the TC-83 strain of VEE virus in the state of Sonora in addition to that

done in 1971 appears to have prevented epizoodemic VEE from advancing

beyond Hermosillo, which is approximately 265 ·kilometers south of the

Arizona-Mexico border (22,23,24,25).

Arthropod vectors and their possible role in the spread of epizoodemic VEE

Epizoodemic strains of VEE have been isolated from at least 25

species of mosquitoes consisting of 7 genera and one subgenus (22,1151

125} (Table 2). Isolation of epizoodemic VEE virus from field-trapped

mosquitoes in itself is insufficient evidence to incriminate a particular·

species as a vector of the disease. Three additional factors must be

evaluated prior to the assignment of vector status (115). They are as

fol lows:

(1) Determination of the infection threshold and infection rate of the mosquito. Infection threshold may be defined as that level of host viremia sufficient to infect 1 to 5 percent of the mosquitoes feeding upon the virus source. Infection ra.te is the percent of mosquitoes that are viremic 14 to 21 days after

8

Table 2. Mosquito species from which epizoodemic VEE viruses have been isolated ( 115,117,123,12~

Mosquito species

Aedes aegypti Aedes angustivittatus Aedes scapularis Aedes serratus Aedes sollicitans Aedes taeniorhynchus

Aedes thelcter

Anopheles aquasalis Anopheles crucians Anopheles neomaculipennis Anopheles pseudopunctipennls. Anopheles punctimacula

Cul ex aikeni i Culex cor.niger Culex niqripalpus Culex guinguefasciatus Culex (Melanoconion) sp.

Culiseta inornata

Deinocerites pseudes

Mansonia indubitans Mansonia tltillans

Psorophora ciliata . Psorophora c i Ii pes

Psorophora confinn is Psorophora discolor

Country

Venezue 1 a Colombia Venezuela Venezuela Texas, U.S.A. Colombia, Venezuela, Costa Rica; Texas, U.S.A. Texas, U.S.A.

Venezuela Texas, U.S.A. Trinidad Texas, U.S.A. Colombia

Colombia Colombia Guatemala Colombia Guatemala; Texas, U.S.A.

Mexico

Costa Rica

Colombia Colombia, Guatemala, Trinidad

Texas, U.S.A • Guatemala Venezuela, Guatemala; Texas, U.S.A. Texas, U.S.A.

9

having fed upon a viremic host. Infection is determined by trituration of each mosquito with subsequent inoculation of one 2- to 4-day-old mouse per mosquito.

(2) Determination of transmission rate of the mosquito. This is obtained by allowing individuals one opportunity to infect a susceptible host .following an incubation period (14-21 days) after the original blood meal. The transmission rate is then calculated by determining the percentage of infected individuals that transmitted the disease agent. Infection is assayed as described ·in (1) after al lowing the arthropod to engorge.

(3) Determination of the extent of mosquito-host interaction. Field studies are employed to define this parameter of the possible vector as determined by (1) and (2). Areas of mosquito-host interaction usually considered are proximity of the host to breeding sites of the mosquito, blood meal identification, abundance of the potential vector species, attraction rate to various sentinel hosts, longevity, and flight range of the mosquito.

Some information on potential epizoodemic VEE virus vectors has been

presented (Tables 3 and 4), but much remains to be done. Simulium spp.

have also been implicated as biologic vectors of VEE virus. However, no

I aboratory data are avai I ab le to substantiate th is hypothesis (16,7D, 121).

The average host viremia needed to infect a suitable ~osquito vector

species with VEE virus is 5.D log1D suckling mouse intracerebral lethal

dose for 5D% of those inoculated per ml (SMICLD50) of viremic ·blood (14,

16) and an average VEE virus inoculum per mosquito bite has been stated 1 to be 3.0 log 10 SMICLD50 For establishment of infection and trans-

mission capability of a mosquito two major barriers must be overcome (17).

When a mosquito feeds upon a host the blood meal moves directly to the

glandular midgut for digestion. To retain the blood meal within the

1chamberlain, R. W., Communicable Disease Center, Atlanta, Georgia. Personal communication. October, 1971.

10

Table 3. Mosquito infection and transmission rates for VEE virus IA (74)

Mosquito species % infected % transmitting

Cy]ex tarsal is 100 100 Aedes triseriatus 100 90 Aedes canadensis 100 50 Psorophora confinnis 96 45 Aedes sol 1 lcitans Bo 44 Aedes aegypti 79 26 Manson i a tit i 11 ans 56 22 Anopheles freeborni 90 22 Mansonia indubltans 72 17 Anopheles guadrimaculatus 16 4 Culex pipiens 12 4

Table 4. Mosquito infection thresholds for VEE virus IB (115)

Mosquito species

Psorophora confinnis Aedes aegvpti Aedes triseriatus Aedes taenlorhynchus Anopheles guadrimaculatus Culex nigripalpus

% mosquitos infected

2 3 2 4 0 0

Vlremic hosts titera

4.9 - 5.2 4.9 - 5.2 6.1 6.7 - 7.1

>6.7 - 7.1 >6.7-7.1

aSuckling mouse lethal does 50"/o per ml (SML050tml) of viremic blood.

11

digestive tract a peritrophic membrane is usually secreted around it

which then serves as a minor barrier to passage of virus from a viremic

blood meal to the midgut epithelial cells. The virus may then attach to

suitable receptor sites if they are ava.ilable. These represent the first

major barrier to infection of the mosquito. Assuming the mosquito is a

suitable host species, the virus adsorbs to the midgut cells and is likely

taken into the cell by the process of pinocytosis or viropexis. Upon

entering the cell it is subjected to the action of a series of enzymes

which uncoat the virion. Replication of the viral nucleic acid and coat

materials then occurs with eventual assembly and maturation of progeny

virions. In the process of maturation the assembled viral nuclear mate-

rial is extruded through the cel 1 's cytoplasmic membrane containing

virus-specified envelope components. Hence, with viral maturation the

progeny virions pass out of the midgut epithelial cell to enter the

hemolymph of the mosquito. Having entered the primitive circulatory

system the virus is then able to infect the organs of the mosquito that

are bathed by the hemolymph. Viral replication ensues, but appears to

continue only in the salivary glands. Within the salivary glands the

virus replicates to high titers where it is considered to persist for

the lifespan of the mosquito. The second major barrier is now at hand.

In order for the mosquito to be infective the virus must be able to bud

out from the acinar cells of the salivary glands into the salivary ducts

or their precursors. If the mosquito in question is a vector species

the virus buds into the salivary gland ducts and the mosquito is then

infective indefinitely according to laboratory studies (16,17). The

12

extrinsic incubation period In the mosquito can be defined as the interval

required for virus to appear in the salivary juices after feeding upon a

viremic host. This period is inversely proportional to the ambient

temperature of the mosquito's habitat. For species of Aedes and

Mansonia the period is probably no more than 8 to 12 days or about-the

span of time between the first and the third blood meals. The period

would probably be even less for a species such as Psorophora confinnis

due to its relatively short lifespan as compared to longer-lived mosqui-

toes such as Aedes sp. (16).

According to the previously listed cri·teria for vector status only

Psorophora confinnis and Aedes taeniorhynchus have been proven as likely

vectors of the IB strains of VEE virus. Insufficient data are available

at this time to unequivocally incriminate these species as vectors of

epizoodemic VEE. Other probable vectors species according to habitat are

as follows (115):

(1) Permanent fresh water with vegetation. Mansonia indubitans and _tl. titillans.

(2) Temporary fresh water pools, sunny and grassy. Psorophora conflnnis, f· discolor, and Aedes thelcter.

(3) Coastal beaches, crab hole terrain. Deinocerites pseudes.

(4) Coastal areas, brackish water, inland 80 to 110 kilometers. Aedes sollicitans and [1. taeniorhynchus.

(5) Woodlands, fresh water, shady pools. Aedes scapularis.

Although the role of mosquitoes as vectors of VEE virus is gradually

being defined, the question of origin of epizoodemics of VEE remains

unknown. Migratory birds have been considered to be a potential means

13

of transport of eastern and western equine encephalomyelitis viruses from

one country to another, but few epizoodemic VEE virus isolations have

been obtained from migratory birds to substantiate such a concept for

VEE virus movement. According to Grayson (51) the only naturally-in-

fected, vlremic (epizoodemic VEE) bird reported has been a fledgling

green heron (Butorldes virescens), also referred to as the striated heron

(B. striatus). On the basis of that isolation Grayson and co-workers (51)

inoculated 9 wild-caught, serologically negative green herons with 100

suckling mouse lntraperitoneal lethal dose 50% (SMIPLD50) of the ~880,

ID (endemic) strain of VEE virus.· The 9 birds were all viremic by the

4th and 5th day postinoculatlon and were used for experimental trans-

mission studies with Deinocerites pseudes as the vector and golden

hamsters as the recipient hosts~ Positive transmission was obtained

indicating a possible role for the green heron in the movement of

epizoodemlc VEE virus strains (IA, IB, IC) from country to country along

the coastal areas as occurred in the epizoodemic that spread from

Guatemala to the United States. Additional supporting evidence is that

green herons commonly inhabit areas where the crab-hole-breeding

Deinocerltes pseudes lives.

To determine If rodents (57,63) could be involved in the main-

tenance of epizoodemlc VEE virus Zarate (135) and Walton (128) inoculated

cotton rats (Sigmodon hlspidus) with epizoodemic strains IC and IB

respectively. With 4 to 1000 plaque-forming units of virus administered

subcutaneously Zarate obtained death patterns of 1 out of 5 and 3 out

of 5 rats inoculated. Walton inoculated 3 weanling~· hispidus with

14

1000 SMICLD50 units of virus and had no survivors by 6 days post-

inoculation. The conflicting results of these two studies and the

statistically small number of experimental animals indicate further

studies in this area are needed to define the possible role rodents

may have· in the maintenance of epizoodemic VEE virus.

Fossaert (40), Franck (41) and Johnson (68) have likewise cited

the possibility of epizoodemic virus residing in natural foci much

as endemic strains of VEE are known to exist. Little evidence other

than that of Grayson (51) and Walton (128) exists to substantiate such

a hypothesis.

Bats have also been proposed as a transport and maintenance mechanism

for epizoodemic VEE by several workers (65). According to Baer, cited

by Calisher (12), the Mexican freetail bat (Tadarida brasiliensls

mexicana) cohabits with the vampire bat (Desmodus rotundus) and

migrates hundreds of kilometers to the United States and to other

regions. In August, 1970, Q. rotundus was found infected with the IB

strain of VEE in the state of Oxaca, Mexico during an epizoodemic (27).

Epizoodemic VEE virus has also been isolated in Ecuador from Q. rotundus

in 1969 (51).

Vampire bats subsist on a strict blood diet and frequently feed upon

equines, consuming as much as 20 to 25 ml of blood per day. While feeding

the bats may either ingest viremic blood or be bitten by infectious

mosquitoes (27). Sanmartin (99) has fed heparinized viremic horse blood

to Q. rotundus and has determined VEE virus to be present in the bat's

oral cavity for!48 to 168 hours postinoculation. Transmission studies

15

have not yet been reported. However, after feeding vampire bats will

often rest in hollow trees which may also serve as resting areas for

mosquitoes. If those mosquitoes were also vector species they could

then feed upon the resting, possibly viremic bats, and later become

Infectious to exacerbate an epizoodemic of VEE (1,29).

Another means of maintenance of epizoodemic strains of VEE virus

that has been postulated is that of silent amplification. Groot (55)

investigated various races of sheep from La Guijara, Colombia and found

that they either developed a transient, minimal viremia or were not

viremlc. However, all of the sheep exhibited serologic conversion from

negative to positive.

Goats have also been considered as possible silent amplifiers of VEE

(57) but confirmatory data are not available at present (104). Serological

surveys conducted during outbreaks of epizoodemic VEE have on the other

hand shown a fairly high ratio of serologic conversion of domestic goats

tested for VEE antibody by the hemagglutination-inhibition and serum-

neutral lzation tests. Bergold (7) found a high incidence of antibody in

goats in Venezuela from 1962 to 1969. By the hemagglutination-inhibition

tests 102 of 332 goats were positive at a serum dilution of 1:20.

Eplzoodemic VEE virus was also obtained from a goat in Zulia, Venezuela

over the eplzoodemlc period of 1968 to 1969.

In an earlier publication, Sellers, et al. (110) described ~ ~

hemagglutlnation-lnhibition and serum-neutralizing antibody titers in

approximately 59 percent of 39 goats sampled from November, 1962 to

January, 1964 in Venezuela. During epizoodemic virus activity in

16

Colombia from 1967 to 1968, Mackenzie (85) obtained an overall antibody

incidence of 20 percent of 31 goats tested. Serum neutralization

antibodies were also found in goats in Texas during the 1971 outbreak

but detailed results are not yet available (94).

The role of the dog as a silent amplifier has.been evaluated by

several groups. Taber, et~· (118) infected beagles with the IA

subtype of VEE virus and obtained adolescent (21-23 day old) mouse

viremia titers ranging from 3.1 to 4.3 log 10 mouse IPLD50 per ml :0f

viremic dog blood. Bivin, et al. (9) were able to transmit IA VEE virus --to beagles with A· triseriatus mosquitoes which had been infected by

the hanging-drop method. Davis, et al. (30) transmitted IA VEE virus --from beagles to guinea pigs with A· triseriatus at viremia titers

ranging from 3.7 to 6.0 log 10 adolescent (21-23 day old) mouse 1PLD50 per ml of blood. However, work done by Sudla (115) indicates

that the threshold for infection of A· triseriatus with the IB

subtype of VEE virus is considerably higher than that indicated by the

work of Davis, et~· (30) with the IA subtype of VEE virus. Based on

the information available it does not seem likely that the dog would

serve as a silent amplifier of IB VEE virus.

Chamberlain (13) has proposed several areas which should be studied

to elucidate the role of alteration of viral virulence in the sudden

eruption of epizoodemic VEE in regions that previously were·11 free" of

v I rus ac:tt vJ t·y. They a re as fo 1 I ows:

(1) Passage in vertebrates with either higher or lower bo~y tempera.tures than the usual hosts.

17

(2) Incubation l.n vectors for either exceedingly long or very short periods.

(3) Passage by the pharyngeal route (abnormal).

(4) Reproductive organ infection and transmission by sexual contact.

(5) Simultaneous infection of either mosquitoes or vertebrates with two strains of virus.

Due to the high magnitude of viremia produced in man by VEE virus

infection he is also subject to consideration as an amplification

mechanism. Viremias in excess of 5.0 .log 10 SMICLD50 per ml of blood

with a r,ange of 3,5 to 6.0 log10 per ml have been reported. Man i·s

therefore a possible vehicle of virus movement but is of secondary

importance when compared to the great mobility of equines (11,36,64,101,

111, 126) •

The movement of viremic equines has very likely played a major role

in the dissemination of epizoodemic VEE in this day of modern, rapid

transportation facilities (103,109,125). McConnell (86) has stated that

about 50 percent of all equines infected by the epizoodemic virus will

be clinically normal. Consequently, frantic owners have been known to

ship their valuable horses out of regions where virus activity is prev-

alent and is responsible for dally reports of equine mortality and

morbid l ty ( 120). Race horses have a 1 so been i ncr im i nated as 1ike1 y

prospects due to their widespread travels from ranches to racetracks (93).

Many factors point to equine movement as a major mode of spread of

epizoodemic VEE virus but little specific evidence has been accumulated

to date.

18

During the 1970 VEE outbreak in Central America an inactivated

vaccine of poor quality was prepared in Nicaragua from IB VEE virus

and distributed for use. Shortly thereafter breaks of the disease

appeared to follow the path of the vaccine. When use of the preparation

was suspended the equine cases of VEE immediately ceased (89).

When 11 new11 epizoodemics of VEE appear the poor monitoring of equine

deaths or complete lack of monitoring must be taken into consideration.

The probability would seem to be great that epi zoodemic virus activity

could have been occurring undetected in isolated regions of a country

with a small but significant number of equine deaths (122) . The con-

current presence of endemic and epi zoodemic VEE viruses in a given

region would present a partially immune equine population and would

likely keep equine disease to a minimum. In this type of situation

the epizoodemic virus activity would probably be detected only on the

periphery of the endemic virus area resulting in a very low level or

complete lack of virus activity being reported.

The mechanism by which the epizoodemic virus overwinters in a given

region is not known. On January 10, 1973, IB VEE virus was isolated

from a pool of 50 Culiseta melanura in Xochimilco, Distrito Federal,

Mexico, the first isolate of an overwintering study initiated in coop-

eration with the Communicable Disease Center (CDC) , Atlanta, Georg i a, by

the Animal Health Department, Ministry of Agriculture and Livestock,

Mexico (22).

19

Young (133) has proposed the phenomenon of recrudescence as a source

of epidemics. Supportive evidence presented was the recurrence of

clinical illness due to the IE subtype of VEE virus in a laboratory field

worker one year after initial illness in a .region of Panama where IE VEE

virus had never been isolated.

Endemic (enzootic) VEE

As listed in Table 1, antigenic groups ID, IE, II, III, and IV are

not epizoodemic strains. Due to their focal nature involving primarily

small rodents and possibly birds they have collectively been denoted as

either endemic or enzootic VEE (4,18,19,46,47,66,72,74,95,107,108,134).

Endemic virus strains have been isolated from 7 species of birds, while

natural antibodies against the virus have been found in at least 23 avian

species. In various laboratory trials all bird species studied developed

low to moderate viremias, usually of 2 or more days• duration. In

mosquito-transmission -experiments, some mosquitoes have become infected

by feeding on birds with viremias as low as 2.6 log10 SMICLD50 of endemic

VEE virus per ml. However, the presence of antibodies against endemic

VEE virus in field-trapped birds has been highly variable. For given

regions of endemic virus activity investigators have reported a complete

lack of serological evidence in field-trapped birds while other groups

have found a very high incidence of endemic VEE virus antibodies in

their field-trapped birds from other endemic regions. Due to the marked

discrepancies at present in this area of study, the role of birds in the

maintenance of endemic VEE virus activity cannot be discounted as

20

insignificant (32,53,54,82,83).

Endemic foci usually do not exceed one qua.rter of a square mile and

are usually found in high rainfall areas with either a tropical or sub-

tropical climate (71). The foci may be in wooded regions and have often

been found to be proximal to fresh-water swamps. The endemic virus cycle

is mosquito-rodent-mosquito with occasional bird hosts comingled as noted

earlier. Marsupials, bats, and raccoons have recently been incriminated

in the cycle with small rodents (8,52,71,80,82,100,102,106). In Florida

the mammals primarily involved in the cycle with Culex (Melanoconion) spp.

appear to be the cotton rat (Sigmodon hispidus), the cotton mouse

(Peromyscus gossypinus), the raccoon (Procyon lotor), and the opossum

(Didelphis marsupialis)(8,15,82). See Table 5 for mammals involved in

the maintenance of endemic VEE virus;

A total of 38 species of mosquitoes in 14 genera and subgenera have

been implicated in the transmission of endemic VEE virus (Table 6).

Among them only 3 species have been reported in the literature as well-

substantiated vectors. Galindo (45) indicated that 2 species of mosquitoes

proven to be efficient natural vectors of endemic VEE virus are Culex

(Melanoconion) aikenii and.£. (tl.) portesi. Mosquito groups other than

Culex (tl.) spp. are probably involved as secondary vectors in the natural

transmission of endemic VEE virus. The third species which has been

clearly implicated as a primary vector of endemic VEE is Culex (tl.)

cedeci (15). These vector species would seem to possess a threshold for

the endemic strains of VEE virus not unlike that of Culex tarsal is and

western equine encephalomyelitis. Naturally-infected rodent species have

21

Table 5. Mammals considered to be involved in the maintenance of endemic VEE virus activity (47' 52, 53,54, 71,80,82,85, 1oo,102, 112, 113, 119, 1 31)

Species Virus Antibody isolation Hla CFb

Rodent Cotton rat

(Si smodon hisEidus} x x Terrestrial rice rat

(Orzom)!S 1 at i ceEs) x x Rice rat

(Orzom)!S Ealustris) x Spiny rat

(Proechlm)!S semisEinosus} x x R Ice rat

(Orzom)!S cali9nosus) x Common rat

(Rattus rattus) x Thorny rat

(HoElomys !J)!mnurus) x Spiny rat

(Proechlm)!S !JU)!annensis or is) x x Rice rat

(Orzomys caEito soeldii) x x Chisel-toothed kangaroo rat

(DiEodom}:'.s microEs) x Cotton mouse

(Perom)!SCUS !JOSS)!Einus) x Forest pocket mouse

(Heterom)!S anomalus} x x Short-tailed cane mouse

(Z)!9odontom)!S brevicauda) x x Deer mouse

(Perom)!Scus maniculatus} x Western harvest mouse

(Reithrodontom)!S me9alotis) x Mouse

(Perom}:'.scus mexicanus) x

aPresence of hemagglutination-inhibition antibodies. bComplement-fixation titers <?: 1: 16. cPresence of serum-neut ra 1 i zing an·t i bodies.

SNc

x

x

Table 5. (Continued)

Species

Marsupial Common opossum

(Didelphis marsupial is) Philander opossum

(Philander sp.) Woolly opossum

(Cal uromys derb I.anus) Murlne opossum

(Marmosa mitis)

Bats --;ii;"t I be us 11 turatus

Artibeus turp is Carollia subrufa Caroll la persplcillata Glossophaga sorcina Artibeus sp. Caroll la sp.

Other.: Raccoon

(Procyon 1 otor) Forest rabbit

(Sylvilagus brasiliensis) Desert cottontail

(Sylvilagus audubonii) Nuttal cottontail

(Syl vi 1 agus nuttal 1 ii) Black-tailed jackrabbit

(Lepus callfornicus) Paca

(Agouti pa ca) Red squirrel

(Sci urus granatens is) Vari~gated squirrel

(Sciurus variegatoides) White-ta i 1 ed ante·l ope squ i rre 1

(Citellus leucurus) Weasel

(Mustella frenata) Kit fox

(Vulpes macrotus)

22

Virus isolation Hla

x x x x x x x x

x x x x

x x x x

x x

x x x

x

Antibody CFb

x x x

x

x

SNc

x

I

.I

23

Table 6. ·Mosquito species from which endemic VEE.viruses have been isolated (2,3,4,15,19,46,47,53172,74,105,113,131,135,136)

Mosquito species

Aedes angustivittatus Aedes atlantlcus Aedes scapularis Aedes serratus Aedes taenlorhynchus

Anopheles aguasalis Anophe 1 es n lmbus Anopheles punctimacula

Coguillettldla (Rhynchotaenla) albicosta

Cogu i 11 ett id i a (Rhynchotaen i a) venezuelensls

Culex corniger Culex coronator Culex nlgrlpalpus Culex guinguefasciatus Culex thriambus Culex (Eubonnea) accelerans Culex (Eubonnea) amazonensls Cu lex· (Me 1 anocon ion) a i ken ii Culex (Melanoconion) albinensis Culex (MelanoC:onion) cedeci Culex (Melanoconion) epanastasis Culex (Melanoconion) iolambdls Cu·lex (Melanoconion) opisthopus Culex (Melanocon.ion) portesi Cu·l ex (Me 1 anocon I on) sp i ss i pes Culex (Melanoconion) taeniopus Culex (Melanoconlon) thriambus Culex (Melanoconlon) vomerifer Culex (Melanoconion) ybarmls

Deinocerltes pseudes

Hemagogus mesodentatus Hemagogus (stegoconops) spegazzinli

Country

Brazil, Colombia, Ven~zuela Florida, U.S.A. Brazil, Mexico, Venezuela Brazil, Trinidad• Venezuela French Gu1ana; Florida, U.S.A.

Brazil, Venezuela Braz i 1 Colombia

French Guiana

Brazil, French Guiana, Trinidad

Colombia Mexico Trinidad; Florida, U.S.A. Brazil, Colombia, Mexico, Panama Mexico Trinidad Trinidad Brazil, Colombia, Panama French Guiana Florida, U.S.A. Trinidad Mexico Mexico Brazil, French Guiana, Trinidad Trinidad Braz 11, French Guiana, Panama, Tr'in i.dad Mexico Brazil, Panama, Trinidad Trinidad

Mexico, Panama

Mexico Braz i 1

Table 6. (Continued)

Mosquito species

Limatus durhami Llmatus fl avisetosus

Mansonla fasciolata Mansonla titillans

Psorophora ferox

Sabeth i n.i sp.

Wyeomyia medioalbipes Wyeomyla mltchelli Wyeoinyla (Dendromy.ia) occulta

24

Trinidad Trin.idad

Mexico Trinidad

Country

Brazil, Mexico, Trinidad

Braz i 1

Trinidad Mexico French Guiana

25

been found to deve.lop vlremlas of 3.9 to 5.0 log 10 SMLD50 per ml of

viremlc blood which persist from 4 to 5 days (45,71). Within endemic

foci vector species have been found to be infected throughout the year

(81). The presence of susceptible host spec.ies for maintenance of the

virus is greatly facilitated by the average lifespan of 6 months for

ground-dwelling rodents. Continuous virus activity is further assisted

by the rodents' selective habit of breeding during the rainy season when

large populations of the vector species are available (71).

The fact that certain species of Culex of the subgenus Melanoconion

such as f.. (tl.) portesi (71) will readily feed upon man presents tne

potential of outbreaks of disease in man due to endemic VEE. Mosquito

species in the genera Aedes, Anopheles, Deinocerites, Mansonia, and

Psorophora are also known to feed avidly on equines and man .. (115).

Since species of these genera are involved in the transmission of both

endemic and ep.izoodemic strains of VEE virus, man is indeed a very 1 ikely

candidate for disease in the presence of high virus activity in a given

region. The first human case of VEE in the United States was due to

endemic virus activity in Florida as reported by Ehrenkranz, et~· in

1968 (35). More recently, however, the epizoodemic of VEE in Texas

resulted in 88 laboratory-confirmed cases of human VEE with al 1 but 2 of

the cases occurring in July, 1971 (24).

The fluorescent antibody .test

The swift spread of epizoodemic VEE and its high morbidity rate

necessitates a rapid di agnost I c system. The fluorescent anti body test

26

(FAT) as developed by Coons, et~· (26) with subsequent modifications

meets nearly all the requirements for a rapid, sensitive diagnostic

test when performed in a tissue culture system.

Several types of globulin separation and purification have been

employed in preparation of fluorescent-antibody conjugates. The two

more commonly employed techniques at present are 50 percent saturated

ammonium sulfate precipitation and chromatographic separation of a

ganvna-globul in-rich (lgG) fra.ction from hyperimmune sera.

Coons,~~· orginally used fluorescein isocyanate for conjugation

(26) but this compound has the disadvantages of being unstable and

dangerous to prepare. The synthesis of fluorescein isothiocyanate (FITC)

by Riggs, et~· in a more stable powder form has essentia.1 ly replaced the

isocyanate compound for use in antibody conjugation (79,97 ,98). · The

labeling of gamma globulin fractions with FITC can be done by either

direct addition or by dialysis labeling. For direct conjugation FITC

Is ordinarily added dropwise to the globulin fraction at a ratio that

has varied from 1:20 (1 mg of FITC to 20 mg of protein) up to 1:200.

Current literature indicates the most suitable range for optimal tagging

to occur Is from 1:100 to 1:200 (44,132).

Dialysis labeling, as described by Clark and Shepard (20), is gener-

ally considered to result in more uniform tagging of the antibody mole-

cules of the globulin preparation. Uniform labeling is also enhanced by

the lack of albumin and any traces of macroglobulins (lgM). If these

proteins are present their affinities for FITC are somewhat greater than

that of the lgG In the globulin preparation resulting in less FITC-tagging

27

of lgG and a final conjugate with high background fluorescence and

decreased specificity of fluorescence (88).

Dilution, tissue powder adsorption, and anion-exchange column

chromatographic purification of conjugates are three methods of removal

or marked reduction of antibody molecules excessively tagged with FITC

after unreacted FITC has been removed by either Sephadex G-25 1 chromato-

graphy or dialysis against a buffered saline solution. Dilution is one

of the most widely used. techniques to reduce.nonspecific staining due

to excessively-tagged antibody molecules and may readily be employed.

with hlgh-titered conjugates. Tissue powder adsorption and anion-

exchange chromatography both reduce the antibody content of conjugates

but they also result in a conjugate that is more specific in its staining

characteristics. The· disadvantage of the tissue powder adsorption

technique is that it can lead to bacterial contamination of the finished

conjugate. The advantage of anion-exchange chromatography is that only

optimally-labeled antibodies are left in the conjugate (28,31,49,87).

1Pharmacia Ltd., Uppsala, Sweden.

28

MATERIALS AND METHODS

Cell cultures

To determine the optimal in vitro cell culture system for the iso-

lation and identification of the IB subtype of VEE virus a compar.ative

propagation study was undertaken. Eight types of ceil cultures were

analyzed for their ability to support growth of the virus with the

production of visible cytopathic effects under a standard nutrient agar

overlay. The cell cultures employed in this study were Vero African

green monkey kidney cell line1, human amnion (FL) cell line1, baby hamster

kidney (BHK-21) eel I 1 ine1, L eel I 1 ine1, goat kidney secondary cel 1

culture2, bovine turbinate cell line2, horse kidney secondary cell culture2,

and duck embryo fibroblast (DEF) primary cell culture. All cell cultures

with the exception of the BHK-21 were grown and maintained with Gibco F-1S3

medium supplemented with 10.0 ml L-glutamate and 10.0 ml sodium pyruvate

per liter of medium plus serum as indicated below. The BHK-21 cell line

was grown and maintained with Stoker's modification4 of Eagle's BME 3 which

is the addition of tryptose phosphate broths to a concentration of 10

percent of the final medium. Antibiotics employed in the two culture

media were 10,000 IU penicillin, 0.13 g streptomycin, and 2.S mg nystatin

per liter. For growth a 10 percent concentration of fetal calf serum

1As obtained from the American Type Culture Collection, Rockville, Md. 2Developed and maintained by Diagnostic Virology, APHIS, USDA, Ames, la. 3Grand Island Biological Co., Grand Island, N.Y. 4stoker, M. 1962. Virology 1S:147-1S1. Soi fco Laboratories, Detro it,. Mi ch.

29

was used, and cells were maintained with medium containing 5 percent fetal

calf serum with the exception of the BHK-21 cell culture which was main-

tained with the growth medium.

For the comparative propagation study each of the 8 cell lines above

was seeded into 25 cm2 Falcon flasks 1 and was inoculated when confluent

with o.1 ml of a tenfold dilution of either cell culture or suckling mouse

brain origin virus. The inoculated flasks wer.e placed in a 37C incubator

for one hour to allow virus adsorption. They were then overlaid with

5.0 ml of 1 percent Noble agar2 containing lx Earles 6553, 0.5 percent

lactalbumin hydrolysate, 0.22 g' sodium bicarbonate per liter, 2 percent

fetal calf serum, 3.3 ml of 1:100 neutral red per 200 ml, and 400 IU

·penicillin, 200 µg streptomycin, and 100 µg nystatin per ml. Plaques

were first counted after 48 hours incubation at 37C and again after an

additional 24 hours incubation at 25C. The endpoint was calculated and

expressed as the number of plaque-forming units per 0.1 ml (PFU/0.1 ml).

Titers were then compared to virus titers obtained by suckling mouse

inoculation to determine the relative sensitivity of each cell culture

to IB VEE virus.

Vi ruses

The GJ9-1BJ strain of VEE virus was isolated from a pool of Psorophora

confinnis mosquitoes collected near Parcelamiento Montufar, Guatemala, in

1Falcon Plastics, Division of BioQuest, Los Angeles, Ca. 2Difco Laboratories, Detroit, Mich. 3Grand Island Biological Co., Grand Island, N. Y.

30

1969, during the course of an epizoodemic (62,116). Working stocks of

this virus were prepared by inoculation of suckling mouse seed virus as

supplied to Diagnostic Virology, APHIS, USDA, Ames, la., by CDC, Atlanta

Ga., into suckling mice and two cell lines, Vero and goat esophagus 1• A

0.5 ml quantity of a 1:100 dilution of 10 percent mouse brain suspension

prepared in 0.01 M, pH 7.2, calcium-and magnesium-free phosphate-buffered

saline containing 0.75 percent bovine serum albumin (BAPBS) was inoculated

into each 75 cm2 Falcon flask of confluent cells. After a one hour

adsorption period at 37C, 25 ml of maintenance medium containing one

percent serum was pipetted into each flask and they were returned to a

37C incubator. Fetal calf serum and normal goat serum were used in the

respective cell cultures at a lower than normal concentration to allow

use of the same stock viruses for tissue culture and goat inoculation

studies. After a 48 hour incubation period the culture fluids were

harvested as stock viruses and were maintained at -70C.

The suckling mice inoculated for stock virus preparation were

observed daily for signs of encephalitis. When 5 to 15 percent of the

suckling mice were either prostrate, moribund, or dead the litters were

harvested, A 10 percent suspension of the pooled mouse brains was

prepared in BAPBS, divided.into 1.0 ml aliquots, and maintained at -70C as

virus stock.

1As obtained from the American Type Culture Collection, Rockville, Md.

31

Experimental animals

Twelve domestic goats of mixed sex from 4 to 18 months of age were

used in the experiments. Prior to purchase the goats were screened by

the hemagglutination-inhibition test and were found to be negative for

antibodies agai·nst eastern equine encephalomyl itis, western equine

encephalomyelitis, and Venezuelan equine encephalomylitis viruses. They

were housed in maximum biological security stalls with a filtered air

intake and exhaust supply system that.is more than 99,6 percent effective

in the removal of viral disease agents.

Albino suckling mice, CF-1 strain, 2 to 4 days of age, obta.ined from

Carworth Farms, Division of Becton, Dickinson and Co., New City, N. Y.,

were used for viremia assays and for titration of virus strains. The

viruses were titrated in tenfold serial dilutions using 8 mice per dilu-

tion. Each mouse was inoculated intracerebrally with 0.02 ml of a given

virus preparation and was then observed daily for 7 days. Moribund,

prostrate, and dead mice were removed and stored at -70C until their

brains could be processed for preparation of·complement fixation (CF) test

antigen to confirm the presence of VEE virus in the affected mice. The

log 10 SMICLD 50 per ml endpoint for each specimen was determined according

to the method of Reed and Muench, cited by Lenette and Schmidt (79).

Serology

Hemagglutination-inhibition (HI) tests were performed by a microtiter

modification of the method of Clarke and Casals (21) in twofold serum

dilutions from 1:10. The test antigen was prepared from GJ9-1BJ VEE

32

virus infected mouse brains by sucrose acetone extraction. Ninety-six

well, u-bottom plastic plates 1 with an arrangement of 8 by 12 wells were

used for the test. A 0.25 percent suspension of goose red blood cells

prepared in dextrose-gelatin-veronal buffer was employed for all hemagglu-

tination-inhibition tests. The mouse brain antigen mentioned above was

then titrated for 4 to 8 hemagglutination units per 0.025 ml using a 0.2%

bovalbumin, pH 6.o borate saline (BBS). Sera to be tested were likewise

diluted in BBS for the HI test. Initially 0.05 ml of a 1:10 serum dilu-

tion was placed in the first well of the plate and 0.025 ml of BBS was

dispensed in the succeeding wells. Twofold dilutions were then made with

0.025 ml microdiluters 1, 0.025 ml of titrated antigen was placed in each

well, and the test was incubated overnight at 4C. The following morning

0.05 ml of a freshly prepared 0.25 percent suspension of goose red blood

cells was added to each test well and the plate was briefly agitated for

mixing of the test reagents. After one hour incubation at 37C the plate

was read for the absence of hemagglutination, indicating a positive HI

test.

Serum virus neutralization (SN) testing was performed in 2 virus-

tissue culture systems. The GJ9-1BJ strain of VEE virus was used with the

BHK-21 cell line and the TC-83 strain of VEE virus was used with DEF cells

for titration of goat sera. All sera tested were heat-inactivated at 56C

for twentymfoutes. The 0.2 ml al lquots of tenfold dilutions of sera being

tested were aseptically dispensed into sterile 12 x 75 mm stoppered tubes

1cooke Engineering Co., Alexandria, Virginia.

33

to each of which were added 0.2 ml of virus containing 120 to 200 PFU

per 0.1 ml. The resultant mixtures were then agitated briefly and placed

in a 37C waterbath for a one hour period of virus neutralization. At

the end of the incubation period the tubes were removed and placed in a

wet ice waterbath. One tenth ml of each mixture was then pipetted into

2 25 cm2 Falcon flasks of either BHK-21 or DEF cells according to the

strain of virus being used in that test. Following a one-hour period of

virus adsorption at 37C, 5 ml of the Noble agar preparation previously

described was dispensed into each flask. The flasks were then placed on

a level surface at 25C for 15 to 25 minutes for agar solidification. When

the agar had hardened the flasks were inverted and were incubated at 37C.

After 48 hours the flasks were removed and the first plaque coun~ was made.

A final count of plaques was made after 24 hours• incubation at 25C. The

endpoint of a serum was that dilution producing at least a 90 percent

plaque reduction.

The complement fixation test was used to confirm that suckling mice

employed for virus titrations which had been either moribund, prostrate,

or dead at the time of harvest were infected with VEE virus. A 10 percent

suspension of mouse brain was prepared in veronal buffer diluent (VBD),

and was centrifuged at 10,000 rpm for 30 minutes. The supernatant fluid

was retained as the 4x CF test antigen. The microtiter CF test' was

performed with either VEE antiserum or mouse ascitic fluid employing

dilutions of 1:8 through 1:256, a 1:20 dilution of unkown antigen, 7

hemolytic units of complement, and sensitized sheep red blood cells. The

34

VEE ant I serum was di 1 uted in the p 1 ates with subsequent add! ti on of

antigen and complement prior.to incubation overnight at 4C for complem~nt

fixation. The following morning sensitized sheep red blood cells were

added and the plate was incubated at 37C for 30 minutes, followed by

centrifugation at approximately 250 g for 10 to 15 minutes to pack the

remaining ghost and red blood cells. The test was read by comparing

hemolysls against known color standards. From zero to 30 percent hemolysis

was considered to be a positive test. Wells displaying greater than 30

percent hemolysls were cons.idered to be negative.

Sentinel study

Six goats were inoculated intradermally with 1000 SMICLD 50 of goat

esophagus cell culture origin GJ9-1BJ VEE virus (GEV). The 6 goats were

divided into 2 groups with one noninfected goat per group to determine if

contact transmission of GEV from goat to goat was possible. Blood samples

were taken with and without heparin from the 8 goats from zero to 14 days

postinoculation (DPI) for determination of viremia and for serologic assay.

An additional aliquot of blood was obtained at zero DPI from each goat to

provide normal goat serum for conjugation. Nasal, oral, and genitourinary

swabs were also obtained 0-7 DPI to determine if VEE virus was shed by

the infected goats. Serum samples were obtained from the 8 goats on a

dally basis from 15-21 DPI and biweekly thereafter through 49 DPI. Rectal

temperatures were recorded dally from 0-21 DPI. Goat viremia assay was

done by BHK-21 plaque assay and intracerebral inoculation of suckling mice.

Serologic responses were determined by HI and SN tests using viruses TC-83

35

and GJ9-1BJ.

Antiserum production

Two groups of goats were employed for the 2 immunization protocols.

Two goats were inoculated subcutaneously (SC) with 1000 SMICLD50 of GEV

In Freund's complete adjuvant 1 at o, 10, and 20 DPI. The 2 goats of the

second group were inoculated SC with 1000 SMICLo50 of GEV, which was

followed by 4 SC injections of the virus-adjuvant mixture described above

at 8, 10, 12, and 18 DPl. Serum samples were obtained at o, 21, 30, 42,

and 49 DPI for antibody assay. By 49 DPI the response of the 4 goats was

not adequate {1:1000) by the SN test with virus GJ9-1BJ and the goats were

subjected to challenge with 25,000 SMICLD50 of GEV intravenously (IV) at

50 DPI. Serologic response was then monitored at 59, 63, 70, 72, and 80

DPI to evaluate the 4 goats' response following challenge. The 2 goats

of the first group displayed the greatest serological response by SN

testing with vi~us GJ9-1BJ at 63 DPI.· Serum neutralization tests were

done in the latter half of each week which meant that the 63 DPI results

were not available until approximately 69 DPI. Serum harvest of the 2

goats by exsanguination was consequently done at 72 DPI. The goats of the

second group were exsanguinated with serum harvest at the termination of

this study at 90 DPI.

Seven of the 8 goats in the sentinel study were also used for the

production of hyperimmune serum. One of the contact transmission goats

was given 40,000 SMICLD50 of GEV IV at 50 DPI and rectal temperature was

1Dlfco Laboratories, Detroit, Mich.

36

taken daily for 7 days. The 6 previously infected goats were challenged

at 50 DPI with 25,000 SMICLD50 GEV given IV. Serologic monitoring was

continued on a biweekly basis through 90 DPI. Exsanguination with serum

harvest of the 7 goats was done at 70 (2 goats), 72 (1 goat), and 90

(4 goats) DPI.

VEE conj ugat_e preparation

The serum of goat 152, which had the highest neutralizing-antibody

titer to VEE using GJ9-1BJ virus, was selected for f.luorescent antibody

(FA) conjugate preparation. The FA conjugate was prepared by 2 different

techniques to assure that one of the conjugates would be of very high

quality. The first technique used was the method described by Goldman (48).

Four m.1 of the goat 152 serum were placed in an Amicon ultrafiltration

ce11 1 equipped with a membrane designed to retain materials of greater

than 100,000 molecular weight. Thirty-six ml of 0.1 M, pH 8 TRIS buffered

saline were added to the cell and the cell was then placed on a magnetic

stirrer set at a low speed to avoid denaturation of serum proteins. A

pressure of 10 psi was applied to the cell for a period of approximately

45 minutes to reduce the mixture to a volume of 4 ml and to remove more

than 80 percent of the serum albumin originally present. The air pressure

was then removed and the fi 1 tered protein so 1 ut ion was app 1 i ed to a 400 cc

co 1 umn packed with Sephadex G-2002equi1 i brated with the TR IS buffer 1 i sted

above. The gamma globulin (lgG) fraction was eluted overni9ht by reverse

1Scientific Systems Division, Amicon Corporation, Lexington, Mass. 2Pharmacia Ltd., Uppsala, Sweden.

37

flow chromatography and was recorded with an ultraviolet scanning device1•

The Lowry technique (84) was used to determine the protein concentration

of the lgG fraction. The lgG fraction was then pressure dialyzed against

0.05 M carbonate.-blcarbonate buffer, pH 9.0, to a 1.5 percent (15.0 mg

protein per ml) concentration and was ·placed in 8.0 mm dialysis membrane

tublng 2• A solution of fluorescein isothiocyanate3 (FITC) equal to 10

times the volume of the lgG fraction was prepared in a 250 ml beaker

with 0.05 M, pH 9.0 carbonate-bicarbonate buffer at an FITC concentration

of 0.1 mg per ml of buffer. The dialysis tubing containing the lgG and a

magnetic stirring bar were placed in the FITC solution at 4C. The beaker

was covered with aluminum foll to prevent evaporation and was stirred

slowly overnight with a magnetic mixer.

The FITC-tagged lgG preparation was then removed from the dialysis

tubing and was placed on a 2,0 x 20.0 cm column of Sephadex G-254 which

was equilibrated with 0,01 M, pH 6.8 phosphate buffered saline (PBS). T~e

conjugate was then eluted with the same buffer and was collected as a

single colored band from the column.

A 2.0 x 20.0 cm column of QAE-Sephadex4 (diethylaminoethyl-Sephadex)

was prepared in 0.01 M, pH 6.8 PBS for purification of the conjugate. The

conjugate was adsorbed to the column and was subsequently eluted by step-

wise addition of 0.1, 0.2, and 0.3 M NaCl to the PBS. Two column bed

1 instrumentat.ion Specialties Co., Lincoln, Nebraska. 2 Union Carbide Corporation, Films-Packaging Division, Chicago, 111. 3 The Sylvana Company, Milburn, N. J, Lot No. 1043. 4 Pharmacia, Ltd,, Uppsala, Sweden.

38

volumes, approximately 130 ml, were collected for each change of buffer.

The fractions were then concentrated to the volume of the original lgG

fraction by pressure dialysis, evaluated for fluorescent antibody activity,

and stored at -20C.

The second technique involved globulin precipitation with anvnonium

sulfate, direct tagging with FITC,·and tissue powder adsorption of the

conjugate (90). A 15.0 ml aliquot of antiserum from goat 152 was frac-

tionated at 4C by dropwise addition of an equal volume of 100 percent

saturated ammonium sulfate solution to obtain a gamma-globulin precipitate.

The resultant solution was stirred overnight at 4C and was then centrifuged

at 250g for twenty minutes. The supernatant fluid was discarded and the

precipitate was dissolved in distilled water to a volume of 15.0 ml. The

protein solution was then reprecipltated twice at 4C over a 3-hour period

as described earlier and the final precipitate was redissolved.in distilled

water to a volume of 8.o ml. The resultant protein solution was diaiyzed

against repeated changes of 0.85% NaCl solution at 4C until free of sulfate

ions (so4) as determined by reacting a portion of the o.85% NaCl solution

with a saturated solution of barium chloride. If so4 ions were present

a white precipitate would have been observed.

The protein concentration of the globulin solution was determined by

the bi uret method (50) using a Co 1 eman spectrophotometer at 540 and 560 m µ..

The protein solution was then adjusted to a concentration of 1% by the

addition of 0.1 M, pH 7.2 PBS (PBS'). The globulin solution was tagged

!

!

39

1 directly at a ratio of 1:20 (0.50 mg FITC per mg globulin). A solution

of FITC equal to one-tenth the volume of the globulin solution was prepared

with 0.5 M, pH 9.0 carbonate-bicarbonate buffer and was added dropwise to

the globulin solutton with constant stirring. The FITC-globulin mixture

was then stirred overnight at 4C with a magnetic mixer.

A 2.0 x 20.0 cm column of Sephadex G-25 equilibrated with PBS' was

used to remove FITC as described for the first conjugation preparation

technique. The conjugate was next adsorbed with acetone-extracted rabbit

liver powder. One g of liver powder was dissolved in 2.5 ml of PBS' for

each 20 ml volume of conjugate. The rabbit liver powder slurry and con•

jugate were then mixed overnight at 4C in a 250 ml beaker with the aid of

a magnetic stirring bar. The following morning the mixture was centrifuged

at 30,000 rpm for hour and the supernatant fluid was retained for further

treatment. After dialysis against PBS• for 60 hours the conjugate was

dispensed Into 1.0 dram screw-cap vials and maintained at -20C •. Conjugate

staining specificity of two conjugate preparations was evaluated by staining

VEE-virus-Infected BHK-21 cells.

Normal conjugate preparation

Four ml of pooled normal goat serum were fractionated by reverse flow

Sephadex G-200 chromatography. The resulting lgG fraction was then con-

jugated according to the first technique described. To obtain a working

dilution the normal conjugate was diluted to equal the intensity of

1 The Sylvana Company, Milburn, N. J. Lot. No. 1043.

40

background fluorescence of the VEE conjugate when both conjugates were

used to stain normal BHK-21 cell sheets.

Specimens for examination by FAT

In addition to FA endpoint titrations of suckling mouse GJ9-1BJ and

DEF TC-83 VEE viruses, 10 percent tissue suspensions were examined by FAT.

The VEE (GJ9-1BJ)-infected equine tissue suspensions were prepared from

spleen, tonsil, liver, salivary gland, pancreas, adrenal gland, thxmus,

lung, cerebellum, renal lymph nodes, splenic lymph nodes, mesenteric

lymph nodes, and prescapular lymph nodes.

Preparation of slides

Baby hamster kidney (BHK-21) cells grown on coverslips in Leighton

tubes were used for al I fluorescent antibody tests (FAT). Four tubes

were employed for each test. When the cells were 85 to 100 percent

confluent the growth medium was decanted and each tube was inoculated with

0.1 ml of the material to be tested. The tubes were then returned to a

37C incubator for a one hour period of adsorption. At that time a 1.25 ml

aliquot of maintenance medium was dispensed Into each tube. Two tubes per

test were decanted and rinsed twice with PBS' at 24 and 48 hours postin-

oculation. The cell monolayers were then fixed at 4C for a minimum of

twenty-four hours by the addition of 10 ml of acetone to each tube.

For staining the fixed BHK-21 coverslips were removed from their

tubes and were allowed to air dry at 25C. Approximately 0.05 ml of VEE

conjugate was applied to each coverslip as a thin film. The cells were

then stained for a minimum of 20 minutes in a moist chamber at 37c. After

41

incubation the coversl ips were rinsed first in PBS and next in distil Jed

water. After drying at 37C, the stained coverslips were mounted"cell

surface down on 2.5 x 7.5 cm clear glass microscope slides with a mounting

medium of 50% glycerin in PBS'.

Two"conjugate specificity controls were employed for FAT, Normal

conjugate was appJ ied to VEE-virus-infected eel I monolayers, and VEE

conjugate was applied to normal cell monolayers. Eastern and western

equine encephalomyelitis virus-infected cell monolayers were also stained

with VEE conjugate to evaluate the conguate•s specificity of staining.

Microscopy

Incident-I ight fluorescence microscopy was used for examination of

fluorescent antibody stained preparations (96). A Leitz Orthoplan

microscope stand with a Leitz fluorescence vertical illuminator and

mercury arc lamp (OSRAM HBO 200) was

system: The excitation filters were

equipped with 1 1 2.0mm KG-1

the following filter

to block the infrared

spectrum, 2 4.0mm BG-38 1 filters to absorb the red spectrum, 2 KP-4902

short-wave pass-interference filters to obtain an excitation wavelength of

490nm, and a 45 degree angle beam splitter TK-510 3 which reflected the

490nm light beam through the objective to the specimen and allowed only

I ight of"= 510nm to be transmitted back to the eyepieces. The barrier

filter system included the beam splitter as described, ~ne TK-515 3 barrier

1schott and Gen., Mainz, Germany. 2Balzars, Vaduz, Lichtenstein. 3E. Leitz, Wetzler, Germany.

42

1 'filter to pass light of :.:515nm, and one K-530. barrier passing light of

::.: 530nm to the eyepieces. The 2 KP-490 filters combined with the TK510

beam spl ltter ,produced a narrow-band filter system with both high.

transmittance (::.: 80%) around 490nm and very low transmittance (:<: 0.0001%)

around 525nm (96).

Fluorescence photomicrography

The Leitz Orthomat fully automatic camera system and Kodak high speed

Ektachrome film2 (ASA rating of 160) were used to record FAT results.

Exposure times ranged from 15 to 45 minutes.

1 E. Leitz, Wetzlar, Germany. 2Kodak, Rochester, N. Y.

43

RESULTS

Comparative propagation of VEE virus

Despite repeated attempts neither cytopathic effect (CPE) or

plaqulng was obtained with the L cell line. Up to 5.0 x 105 SMICLD50 of

the GJ9-1BJ strain of VEE virus were used in combination with a moist

37C 5% co2 Incubation system as employed by Hardy and Brown (60). In a

similar fashion, repeated attempts failed to produce either CPE or

plaquing in goat kidney, human amnion (FL), bovine turbinate, and horse

kidney cell cultures.

The Vero African green monkey, duck embryo, and BHK-21 cell cultures

were determined to be of approximately equal sensitivity in the propagation

of virulent VEE virus. Cytopathic effects (CPE). were not readily apparent

at 24 hours postinoculatlon under agar overlay. By 48 hours postinoculation

CPE were evident and were somewhat different for each of the 3 cell

cultures (Table 7).

The CPE of the Vero .ce 11 1 i ne consisted of 70 to 90 percent cytolysis·

of the plaque regions with rounding up of the remaining cells. Plaques

became more difficult to detect between the 146th and 150th cell passages

and the cells essentially lost their sensitivity to VEE-virus-induced CPE

after 150 to 155 passages.

Duck embryo cells seemed to be more resistant than the other 2 cell

types to VEE-induced cytolysis. As few as 5 percent and a maximum of 60

percent of the eel ls· within a plaque were lysed due to virus replication

with 20 to 95 percent of the remaining cells rounding up. Rounded up and

morphologically normal cells also had very fine cytoplasmic vacuoles

44

Table 7. Susceptibility of different cell types to GJ9-lBJ VEE virus

Ce 11 Mean titer Suck I ing Cytopathic Mean plaque culturea effectsb culture

Vero

Duck embryo

L eel I

Goat kidney

Human amnion

Bovine turbinate

BHK-21

Horse k I dney

a PFU/ml.

cell

6.4 x 108

2.7 x 108

Negative

.Negative

Negative

Negative

4. 3 x 10 8

Negative

mouse ICLD50

5 x 1010 48 hr, 70-90%

5 x 1010 48 hr, 5-60%

5 x 1010 ------9.1 x 10 10 ------9. 1 x 1010 ------9. 1 x 1010 ------

5 x 1010 48 hr, 30-75%

9. 1 x 1010 ------

bHours postlnoculation, % cells affected in the plaques. cDetermined during final 72 hr postinoculation plaque count.

diameterc

3.7mm

2.7mm

-------------------------3.3mm

------

45

present.

The overall cytopathic effect on BHK-21 cells was not as marked as

that se.en in Vero cells. Cytolysis varied from 10 to 45 percent of the

plaque cell population with 5 to 75 percent rounding up. Cytoplasmic

vacuolization was more marked in BHK-21 cells than that observed in duck

embryo cells and was usually observed in greater than 30 percent of the

plaque cells.

The goat as a sentinel

Serum neutralization (SN, TC-83 VEE virus) antibody titers of 1:10

or greater were detected in of the 6 infected goats at 6 days postin-

oculation (DPI), 2 goats at 7 DPI, 5 goats at 8 DPI, and all goats by 9

DPI. The SN titers in 5 of the 6 goats rose very rapidly and by the

second day of response had increased from 1:10 to 1:100 (Figure 1).

Serum-neutralization testing of the sera with GJ9-1BJ VEE virus did

not detect a serological response until 8 DPI in 1 goat. By 10 DPI 5 of

the 6 goats had 1: 10 titers. The 6th goat seroconverted by 15 DP I, was

negative at 1: 10 for the next 3 days, and regained a 1: lO titer at 19 DPI.

The 5 goats with 1:10 titers at 10 DPI responded in a serologically uniform

manner for the remainder of the 45 day monitoring period (Figure 2).

Maximal SN response with GJ9-1BJ virus was 1:100 in 1 goat as compared

with TC-83 virus SN titers of 1:1600 in 3 goats, and 1:800 in the other 3

goats of the group (Figure 1).

Hemagglutination-inhibition (HI) titers did not appear until 7 DPI

(1 goat). The maximal HI response was 1: 10,240 in 1 goat but another I

Figure 1. Senti·nel goat study. TC-83·SN antibody titers of goats 147, 152, 157, 158, 159, and· 161 between days 6 and 45 postinoculation. Titers listed were the highest serum di 1 ut ion that produced at 1 east 90% p 1 aque reduction'. See Append i'x for specific titers.

47

147 152

157 158

2560 159 161 128 640 020 160

80

40

"' ... 20 I-;:

10

<10

6 16 26 36 45 DAYS POSTINOCULATION

\

Figure 2. Sentinel goat study. GJ9-1BJ SN antibody titers of goats 147, 152, 157, 158, 159, and 161 between days 6 and 45 postinoculati-on. Titers listed were the highest serum dilution that produced at least 90% plaque reduction. See Appendix for specific titers.

50

goat did not exceed a titer of 1:40 during the observation period (Figure

3). The 2 contact 'transmission goats did not display detectable VEE viral

antibodies by the 3 serological assay methods employed in this study.

(See Appendix for titers by HI and SN).

The goat as a silent amplifier of IB VEE virus

Viremia was not detected by the plaque assay technique using BHK-21

cells,, By suckling mouse inoculation 5 of the inoculated goats were

found to be viremic for 1 to 3 days, viremia commencing between the 1st

and 5th days postinoculation. One of these goats was viremic from day 1

to day 3 postinoculation, another from day 2 to day 4, and a 3rd from day

3 to day 4. One goat was viremic on days 3 and 5, and one goat was viremic

only on day 4. Peak viremia did not exceed 4.08 log 10 SMICLD50 per ml of serum. Respective SMICLo50tml of the sera are listed in Table 8.

Oral, nasal, and genitourinary swabs obtained from day zero through day 7

were negative for VEE virus by suckling mouse inoculation.

Clinical evidence of disease in the domestic goat was lacking (Figure

4) during the 21-day observation period with one exception. Goat number

159 displayed an increased body temperature from day 2 through day 10,

which peaked on day 5 at 41.1C (105.9F), The goat 1 s normal body temperature

was 39.lC (102.3F) as determined from baseline data (Table 9),

VEE antiserum production

After intravenous challenge of the 3 groups of goats at 50 days post·

inoculation, the maximum antibody titers as determined by the 3 tests were

Figure 3.

)

Sentinel goat study. HI antibody titers of goats 147, 152, 157, 158, 159 and 161 between days 6 and 45 postinoculation. See Appendix for specific titers.

'

~ w ~

~

52

157

!5120 159

2!560 1280 640 320 160

60

40

20

10

<10

6 16 26 :56 4!5 DAYS POSTINOCULATION

152

108

161

53

Table 8. Virus titers in serum of goats inoculated with GJ9•1BJ VEE virus

Goat number Days Postinoculation Magnitude of Viremia (log 10 SMICLD50tml serum)

147 1 4.08 2 2.2 3 2.2

152 0-14 o.o 157 3 < 1. 7

4 < 1. 7 158 4 1.95 159 2 < 1.7

3 3.27 4 < 1. 7

161 3 2. 36 5 < 1. 7

Table 9. Body temperatures of goat number 159 inoculated with IB VEE virus

Days postinoculation Body temperature

0 1 2 3 4 5 6 7 8 9

10 11 12

Normal body temperature

C F

39.2 38.9 39. 1 39.6 40.7 41.1 40.2 40.2 40.3 40.9 40.1 38.7 38.9 39. 1

102.5 102.1 io2.4 103.2 105.2 105.9 104.4 1.04. 3 104.6 105,3 104. 2 101.6 102. 1 102.3

54

1:51,200 (SN~ TC-83, 7 of 12 goats), 1:1280 (SN, GJ9-1BJ 2 of 12 goats),

and 1:64,000 (HI, 5 of 12 goats). See Appendix for listing of titers from

zero through 90 days postinoculation. Serum from goat number 152 exan-

guinated at 72 DPI was selected for conjugation on the basis of a

GJ9~1BJ SN titer of 1:1000.

Conjugate evaluation

After the QAE-Sephadex conjugate fractions had been concentrated by

pressure dialysis they were examined for specific fluorescence on TC-83

VEE virus-infected BHK-21 cells at 24 and 48 hours postinoculation. Only

that fraction obtained by addition of 0.2 M NaCl demonstrated specific

fluorescence. The fluorescence was finely granular in appearance and

occurred only in the cytoplasm of infected cells (Figures 8, 10, and 11).

Specificity of staining was evaluated by 2 methods. Baby hamster

kidney (BHK-21) cells were infected with approximat~ly 103 PFU per ml

of eastern equine encephalomyelitis or western equine encephalomyelitis

viruses. At 24 hours postinoculation the infected cell cultures were

fixed in acetone at 4C and were examined 1 day later with the conjugate.

Cross-staining resulting in unwanted specific fluorescence did not occur

(Figures 12 and 13).

The second conjugate specificity parameter was the staining of normal

BHK-21 cells 24 hours after they had been subjected to the same regimen as

that employed for infecting cells. Nonspecific staining of normal cells

was not observed (Figure 5).

Ffgure 4. Absence of clinical signs in a domestic goat three days after infectfon with the GJ9-1BJ straf n of Venezuelan equine encephalomyelltis (VEE) virus.

Figure 5. VEE conjugate applied to normal BHK-21 cell monolayer. Note absence of specific fluorescence. 125x

r

56

Figure 6. VEE conjugate applied to GJ9-1BJ VEE-virus-infected BHK-21 cell monolayer at 24 hours postinoculation. Note specific perinuclear and cytoplasmic fluoresence. 125x

Figure 7. Normal conjugate applied to GJ9-1BJ VEE-virus-infected BHK-21 cell monolayer at 24 hours postinoculation. Note absence of specific fluorescence. 125x

58

Ffgure 8. VEE conjugate applied to TC-83-VEE-virus-infected BHK-21 cell monolayer at 24 hours posti nocul ation. Note specific, granular perlnuclear and cytoplasmic fluorescence. 500x

Figure 9. VEE conjugate app lied to GJ9-1BJ-VEE-virus-infected BHK-21 cell mono l ayer at 24 hours postinoculation. Note specific, granular perinuclear f luorescence. 1188x

60

Figure 10. VEE conjugate applied to TC-83-VEE-virus-infected BHK-21 cell monolayer at 48 hours postinoculation. Note spec i fic cytoplasmic fluorescence of rounded up cells. 125x

Figure 11. VEE conjugate applied to TC-83-VEE-virus-infected BHK-21 cell monolayer at 48 hours postinoculation. Note specific, granular cytoplasmic fluorescence of rounded up cells. SOOx

62

- ..... .. ... '· .

:-.. . • .. !...r • . •1 .. .

~

·~"' • ... . ~ .. t •" I 4 ,.

.:t. ..,, # " • •

• • .. "" ...

' I 9 ,. • • :-• ~ '

~· • t

' • • ' ' • ,,,, ·. . • ,, ., .\ I! ' ). ' • • • ; •

;~. "' ' "' ' • ,, •,

4t I

• • ,..

Figure 12.

Figure 13.

VEE conjugate applied to western equine encephalomyelitis virus-infected BHK-21 cell monolayer at 24 hours postin-oculation. Note absence of cross-sta ining. 125x

VEE conjugate applied to eastern equine encephalomyelitis virus-infected BHK-21 cell monolayer at 24 hours postin-oculatton. Note absence of cross-staining. 125x

64

65

When the direct 1 y-tagged conjugate prepa_rat ion was used to stain

TC-83 virus-infected BHK-21 cells marked nonspecific staining was

observed. Dilution of the conjugate beyond 1:8 was not feasible due to

markedly diminished specific staining and at the 1:8 dilution the level

of nonspecific fluorescence was not tolerable. On that basis further

fluorescent antibody studies on VEE virus were done with Q.AE-Sephadex-

treated conjugate.

The n<irma 1 -goat serum conjugate was prepared from Sephadex G-200

fractionated lgG which was tagged with FITC and purified by Q.AE-

Sephadex column chromatography. The working dilution of 1:4 was

derived by colll'arison of background fluorescence of both the normal

and VEE conjugates when VEE virus-infected and normal BHK-21 cell

monolayers were stained (Figures 5, 6, and 7). This conjugate

was then employed as a specificity of staining control when conducting

fluorescent antibody tests (FAT) on VEE virus-infected pony tissue

suspensions.

Comparison of sensitivity of FAT to suckling

mouse inoculation for virus identification

Initially the 2 stock virus preparations employed in this study were

assaye_d by the fluorescent antibody test and intracerebral inoculation of

suckling mice. Fluorescent antiboqy assay in BHK-21 cells was found to

have approximately 1.0 log 10 per ml greater sensitivity.

When 20 viremic pony tissues were tested by the 2 systems suckling

mice were the most sensitive virus detection system. The threshold of

66

sensitivity of the fluorescent antibody test appeared to be between 1.6

and 2.0 log 10 SMICLD50/ml of 10 percent pony t.issue suspension. By

FAT all tissue suspensions were positive by 24 hours postinoculation

with the exception of 4 of 6 tissues in the 2.0 to 2.8 log10 SMICLD501ml

virus titer range which were positive at 48 hours postinoculation. Two

tissues with a titer of 1.6 log 10 SMICLD50tml were negative at 24 and 48

hours postlnoculatlon.by the fluorescent antibody test (Table 10).

Table 10. Virus detection by the fluorescent antibody test (FAT) and by intracerebral inoculation of suckling mice

Specimen . a Pony number

TC-83 virus 10% suckling

mouse brain GJ9-1BJ virus

Spleen 18 Tons i 1 18 Renal and splenlc 19

lymph nodes Adrenal 19 Salivary gland 19 Liver 19 Spleen 522 Adrenal 522 Mesenteric 522

lymph node Liver 522 Thymus 522 Renal lymph 522

node Pancreas 522 Lung 524 Spleen 524 Thymus 526 Mesenteric 526

lymph node Cerebellum 529 Prescapular 530

lymph node Sp 1 een 530

FATb Hours post inoculation

> 9.0 24 :i:10.o 24

Pos. 48 Pas, 24 Pos, 24

Pos. 24 Pos, 24 Pas. 24 Pos. 24 Pas. 48 Pas. 24

Pos. 48 Pos. 24 Pas. 24

Pas. 24 Neg. 48 Pas. 24 Pos. 24 Pas. 24

Neg. 48 Pas. 24

Pos. 48

b SMI CLo50

8.7 9,5

2. 1 2. 1 7. 1

6.3 6.o 6.7 3,5 2.8 5,7

3,6 5,2 3.8

4.o 1. 6 2.2 5,3 6.8

1. 6 4.3

2.0

with aTen percent tissue suspension in 0.753 BAPBS from ponies inoculated 1000 SMI CLD50/ml GJ9-1BJ VEE virus intradermal ly. bLog10 per ml of inoculum; positive or negative (FAT).

68

DIS CUSS I ON

The baby hamster kidney (BHK-21) cell line has been proven superior

to either primary duck embryo or Vero African green monkey cell lines for

titration of Venezuelan equine encephalomyelitis (VEE) virus. The Vero

cell line is slightly more sensitive for the titration of the GJ9-1BJ

strain of VEE virus but It lacks the potential for continuous serial

passage that the BHK-21 line has as supplied from the American Type

Culture Collection. Baby hamster kidney cells have been passaged in

excess of 200 times at the National Animal Disease Laboratory, Ames, Iowa,

without loss of sensitivity for VEE virus. In contrast, the Vero cell

line is supplied at the 122nd passage level and has been found in this

laboratory to lose its susceptibility to VEE virus between the 150th

and 155th passage level. Earley, et~· (34) also did not find Vero cells

to be sufficiently susceptible after 30 serial passages. Thus, the time

saved by not having to restart the cell line from the 122nd passage level

every three months more than justifies the choice of the BHK-21 cell line

over the Vero cell 1 ine.

Primary duck embryo cell cultures are slightly less sensitive for the

detection of the epizoodemlc strain of VEE virus than BHK-21 cells. The

mean VEE plaque diameter in the duck cells is also 1.0 mm less than in

BHK-21 cells which makes enumeration of plaque-forming units more difficult.

The BHK-21 cell line is thus the cell culture of choice of the 8 evaluated

in this study.

Experimental lnoC:u.lation of the goat with the minimal exposure of

1000 SMICLD 50, an average mosquito inoculum, revealed the goat to be·an

excellent sentinel for VEE virus activity. The 6 inoculated goats

developed titers greater than 1:10 as determined by hemagglutionation-

inhlbltlon (HI) and serum neutralization (SN) tests. Both persisted in

excess of 40 days. In view of these findings the investigation of

Dickerman, ~ ~· (33) would seem to have provided questionable information

on the bovine as a possible sentinel species. In that· study 8 native HI

and SN seronegative Colombian bovines were inoculated with a great excess

of IB VEE virus, 7,1 log 10 primary chicken embryo cell. plaque-forming

units (CEC-PFU), to "insure establishment of infection." The 7.1 log1·0 CEC-PFU of virus would be roughly equivalent to 9 log 10 SMICLD50 •

Considering an average mosquito inoculum of 1000 SMICLD50, approximately

1,000,000 average IB VEE viremic mosquitoes would have had to feed upon ' a single animal over a given period. In view of the Information obtai.ned

herei.n with another domestic ruminant species, the goat, it would appear

necessary to repeat that study with a lower inoculum of IB VEE virus.

However, sufficient numbers of goats must be available to the

potential vector to be able to detect VEE virus activity in an area.

Hayes, et ~· (61) have demonstrated in a moquito host preference study

in Hale County, Texas, that the predominant feeding habit of mosquitoes

of the genera Aedes, Anopheles, Culiseta, and Psorophora is to feed

upon mammalian species. Sudla (115) has stated that only Psorophora

confinnis and Aedes taeniorhynchus have been proven as possible vectors

70

of the IB strain of VEE virus. He has also reported that 83 virus

isolates compatible with VEE were obtained from Psorophora confinnis

during the Texas epizoodemic (117). The bionomics off· confinnis have

been shown to be associated with temporary fresh water pools located in

sunny, grassy areas (115). Such areas can often be found in livestock

pastures. Therefore, in regions where large goat populations are present

the likelihood of the goat being a-sentinel of epizoodemic VEE virus

activity would appear to be very good.

Viremic goats on the other hand, do not offer a source of silent

amplification of epizoodemic VEE virus according to the results of this

study. Goat viremia did not exceed 4.08 log 10 SMICLD50!ml, with a mean

peak viremia of 2.67 log10 SMICLD50/ml. The average viremia required to

infect vector species of mosquitoes has been stated by Chamberlain (14,

16) to be 5.0 log 10 SMICi.o50/ml. Experimentally the Infection threshold

of Psorophora confinnis has been found to be 4.9 to 5.2 log10 SMICLD50/ml

(115). Consequently, the goat is not likely to be an amplification

mechanism of ep i zoodem i c VEE virus.

Due to the excellent serologic response of the goats inoculated as

sentinels they were hyperimmunized in an attempt to produce a more specific

VEE antiserum than that which Was likely produced by the goats given

multiple virus injections to stimulate antibody formation. The sero-

logical response of the 4 antiserum goats was no better than that of the

goats of the sentinel study given only one injection of virus. The serum

of goat number 152 was chosen for conjugate preparation because of its

high.antibody titer as determined by the GJ9-1BJ SN test. Serological

71

testing by the other two techniques, HI and TC-83 SN later revealed an

obvious lack of correlation of serum titers. The most plausible

explanation of such differences is the presence of residual infectivity

representing nonneutralized virus. Several workers have reported a

similar phenomenon of nonneutralized virus in other virus-serum

neutralization test systems. Ashe and Notkins (5), Bradish, et ~· (10),

and Wall is and Melnick (127) have theorized residual infectivity to be

due to the formation of infectious virus antibody complexes which resist

further neutralization by antibody. Lafferty (78) used kinetic serum

neutralization testing to show that residual infectivity was due to

lack of antibody avidity. Fazekas 'de St. Groth, et~· (38) set forth

the concept that residual infectivity was due to dissociation of the

virus-antibody complex. Through extensive kinetic serum neutralization

testing with VEE virus Hahon (58,59) has found that resid.ual infectivity

of VEE virus is not due to dissociation of noninfective antigen-antibody

complexes, to the presence of a genetically stable persistent viral

subpopulation, or to the presence of free infectious ribonucleic acid in

the virus preparation used for the serum neutralization test. His

experiments with TC-'83 and Trinidad IA strains of VEE virus (58) have

clearly demonstrated 2 factors contributing to VEE virus SN residual

infectivity. One factor is the formation of viral aggregates of the virus

preparation employed in the test system resulting in incomplete neutraliza-,

tion. Hahon•s main concept was, however, that residual infectivity was

due to the presence of sensitized VEE virions that were not neutralized '.

72

by the antiserum employed in the SN test. Supporting evidence for such a

theory was obtained when neutralized suspensions were incubat~d at 35C

for 1 hour with a 1: 10 dilution of anti-lgG serum. Addition of the anti-

lgG serum to a neutralized suspension yet containing 5.4 log10 units of

residual virulent IA VEE virus infectivity further reduced viral in-

fectivity of the suspension by an additional 3.7 log10 units. Similar

work has al!\o been done with lactic dehydrogenase virus and pol iovirus

(73,91,92).

A third component of Hahon's study was the determination of residual

infectivity of Trinidad IA VEE virus as compared to the TC-83 attenuated

strain. The percentage of original lnfectivity remaining after serum

neutralization with a 1:50 dilution' of monkey VEE antiserum to Trinidad

IA VEE virus was approximately 20% as compared to 2.2"/o residual activity

for TC-83 VEE virus after 15 minutes incubation at 35C. Clearly, the low

titers obtained with. the GJ9'-1BJ SN test as compared to those of the

TC-83 SN tests would·appear to have been the result of residual infectivity

of the virulent IS.strain of virus. The minor amount of residual infec-

tivity of the TC-83 SN test system made it possible to obtain good

correlation with the VEE HI test. The information subsequently derived

from the TC-83 SN and VEE HI tests indicated that several goats could

have served as .a source for high-ti tered anti sera for conjugation.

The fluorescent antibody conjugate prepared from the serum harvested

from goat number 152 22 days after intravenous challenge With 25,000

73

SMICL050 of GJ9-1BJ VEE v.irus was determined to b.e a highly specific

preparation. Although antigenic interrelationships with eastern equine

encephalomyel it is (EEE) and western equine encephalomyel it is (WEE)

viruses were detected by EEE and WEE HI tests, they could not be demon-

strated with the fluorescent antibody test.

Pooled normal goat serum was used to prepare a normal serum conjugate

for use as a VEE conjugate specificity of staining controi. When the VEE

conjugate was used on the BHK-21 cell line in a fluorescent antibody test,

as little virus as 2.1 log10 SMICLD50to.1 ml inoculum from GJ9-1BJ VEE-

virus-infected .pony tissue suspensions could be detected. If the Leighton

tube inoculum would have been increased to 1.0 ml of tissue suspension·

the sensitivity of the VEE fluorescent antibody test (FAT) would have

likely been within 1 log10 dilution of that of intracerebral inoculation

of 2- to 4-day-old suckling mice. The smaller inoculum was chosen for

use in FAT to make it a more rapid diagnostic test. If the 1 arger

inoculum·had been employed at least two washings with tissue culture

medium would have been required to remove toxic tissue components from

the BHK-21 monolayer after the 1 hour virus adsorption period, An

additional factor to be considered was that of personnel safety. The

use of a smaller inoculum with no washing of monolayers decreased the

possibility of aerosol exposure to laboratory personnel.

74

SUMMARY

The baby hamster kidney (BHK-21) cell line was shown to be superior

to either primary duck embryo cells or Vero African green monkey cells

as a host system for propagation of the epizoodemi·c strain, GJ9-1BJ, of

Venezuelan equine. encephalomyelitis (VEE) virus. For that reason the

BHK-21 cell line was chosen for evaluation of VEE fluorescent-antibody

conjugates.

Surveillance of possible VEE virus activity is of critical importance

in avoidance of major epizoodemics. The goat has been evaluated as a

sentinel for IB VEE virus activity in this study and was found to be a

very sensitive sentinel animal. Six goats of mixed sex from 4 to ·18

months of age when exposed to a single average mosquito inoculum of VEE .. . virus began to seroconvert within 6 days. Serological evaluation using

the TC-83 and GJ9-1BJ serum neutralization tests and the VEE hemagglutina:..

tion-inhibition test showed that the 6 goats were seropositive for VEE

antibody for 49 days. A minimum of VEE virus thus stimulated a very

adequate serological response in the goat.

Following challenge of the sentinel goats with 25,000 SMICLo50 of

the GJ9-1BJ strain of VEE virus a suitable serum for conjugation was

obtained. The VEE antiserum and normal goat serums were conjugated and

evaluated for staining specificity in the BHK-21 cell line. No cross-

staining of the VEE conjugate with eastern equine encephalomyel it is virus

or western equine encephalomyelltis virus was observed despite a 1:10

cross reaction for both vi r.uses as detected by EEE and WEE hemagglutina-

tion-inhibition testing of the same serum.

75

The conjugate was assayed for sensitivity for the detection of VEE

virus in cell cultures inoculated with equine tissue suspensions. The

(BHK-21) cell line when combined· with a conjugate highly specific for VEE

virus has been proven to be nearly as sensitive as intracerebral inoculation

of the suckling mouse for the detection or identification of VEE virus.

In a diagnostic sense the test can now be applied to routine screening of

VEE diagnostic specimens. All negative specimens should then be tested in

suckling mice by the intracerebral route for isolation of VEE virus not

detected by the fluorescent antibody test. The fluorescent ant.ibody test·

wou.ld be of greatest value in the event of an another outbreak of

eplzoodemic VEE among the equidae of the United States. Large numbers

of specimens likely to be positive for VEE virus would be encountered and

a rapid, efficient technique such as the fluorescent antibody test would

then be of critical importance.

76

APPENDIX

TRIS Buffered Saline pH 8.0

1. O. SM TR IS:

Dissolve 60.57 g TRIS and 4.o g sodium azide (NaN3) in 200 ml triple-distilled, deionized water and q,s. to 1000 ml.

2. o.sM HCl:

q.s. 42 ml of 11.9 M HCl to 1000 ml.

3. Buffer preparation:

a. two parts TRIS (133.3 ml) and one part HCI (66.6 ml) are mixed.

b. add. 1.19 g of NaCl to 200 ml of TRIS buffered saline for a final NaCl concentration of O.lM.

c. add 4.0 g NaN 3 per liter of final buffer.

77

0.5 M Carbonate-Bicarbonate Buffer

1 • Reagents required:

a, 0.5 M solution of sodium bicarbonate (NaHC0 3)

b, 0.5 M solution of sodium carbonate (Na2co3)

2. Mix 50 volumes reagent a with 4.5 volumes reagent b (pH should be 9.0 when di luted 10 times in triple-disti 1 led water},

78

0,1 M Phosphate-Buffered .Saline

pH 7. 2:

1. Prepare stock solutions a and b:

a, 0.2 M monobasic sodium phosphate (NaH2Po4) Dissolve 27.8 g NaH 2Po4 in 1000 ml distilled water.

b. 0.2 M dibaslc sodium phosphate (Na2HP04) Dissolve 53.65 g Na2HP04 · 7H2o in 1000 ml distilled water.

2. Mix 28.0 ml of Solution a with 72.0 ml of solution b. Di 1 ute to a total of 200 ml with distilled water.

pH 6.8:

1. Prepare stock solutions a and b as· shown.

2. Mix 51.0 ml of solution a with 49.0 ml of solution b. Di 1 ute to a total of 200 ml with distil led water.

79

Dextrose-Gelatin-Verona! Buffer

1. Dissolve 0.58 g 5,5-diethyl barbiturate acid and 0.6 g gelatin in 250 ml distilled water by heating.

2. Combine the following reagents with solution 1:

Sodium 5,5-diethyl-barbiturate CaC1 2 (anhydrous) MgS04 • 7H 20 NaCl Dextrose

3. q.s. to 1000 ml with distilled water.

0.38 g 0.02 g 0.12 g 8.50 g

10.00 g

\

80

0.2% Bovalbumin, pH 6.o Borate Saline (BBS)

1. Prepare pH 9, 0 borate saline as fol lows:

a. mix 80 ml of 1.5 M NaCl, 100 ml of 0.5 M H3Bo3, and 24.0 ml of 1.0 M NaOH.

b. q,s. to 1000 ml with distilled water.

2, Add Cohn fraction V bovine serum albumin to borate saline (1) to a concentration of o.4%.

3. Prepare 0.15 M NaCl-0.2 M Na 2HP04 as follows:

a. mix 100 ml of 1.5 M NaCl and 100 ml of 2.0 M Na 2HP04 b. q,s, to 1000 ml with distilled water.

4. Prepare 0.15 M NaCJ-0.2 M NaH/04 as fol lows:

a, mix 100 ml of 1.5 M NaCl and 100 ml of 2.0 M NaH2Po4 b. q.s. to 1000 ml with disti 1 led water.

5, Mix 11 ml of solution 3 with 89 ml of solution 4.

6. · Mix equal volumes of solutions 2 and 5, check pH, and BBS is then ready for use.

81

Veronal Buffer (VBD)

1. Dissolve 42.5 g NaCl ahd 1.87 g sodium 5,5-diethyl-barbiturate · in 700 ml distilled water.

2. Dissolve 2.875 g 5,5-diethyl barbiturate acid in 250 ml hot distilled water.

3. Dissolve 20,333 g MgC1 2 (1.0 M) and 4.411 g CaC1 2 (0.3 M) in 1000 ml distil led water.

4. 5 x VBD:

a. Mix solutions 1 and 2 and cool to room temperature.

b. Add 2.5 ml of solution 3 to a and q.s. to 1000 ml with distilled water.

c. To use, di 1 ute 1: 5 and add 1.0 g gelatin per 1000 ml of final diluent.

82

Sentinel goat serological response·- Hla

DPI b Goat Number 147 152 157 158 159 161

0 < 10 <10 <10 <10 <10 <10 1 < 10 <10 <10 <10 <10 <10 2 <10 <10 <10 <10 <10 <10 3 <10 <10 <10 <10 <10 < 10 4 <10 <10 <10 <10 <10 <10 5 <10 <10 <10 <10 <10 <10 6 <10 < 10 <10 <10 <10 <10 7 20 <10 <10 <10 < 10 <10 8 640 <10 10 <10 10 10 9 5120 10 160 <10 20 20

10 2560 20 320 10 80 160 11 2560 20 320 20 320 320 12 1280 40 640 40 320 320 13 1280 40 640 320 320 320 14 640 40 640 2560 2560 640 15 2560 40 1280 2560 1280 640 16 1280 40 2560 2560 640 320 17 640 20 2560 1280 320 640 18 1280 20 640 5120 640 1280 19 640 20 640 5120 640 1280 20 1280 20 320 2560 320 640 21 5120 20 320 2560 320 64o 24 2560 10 320 2560 320 1280 30 5120 10 2560 5120 64o 2560 35 10,240 <10 640 2560 1280 1280 38 2560 < 10 . 640 1280 640 1280 42 5120 <10 640 1280 1280 640 45 1280 < 10 640 640 2560 2560

aReciprocal of highest serum dilution giving inhibition of hemaggluti-nation.

bDay postinoculation.

I I

·.I !

I

83

Sentinel goat serological response - SN(GJ9-1BJ)a

DP lb Goat Number 147 152 157 158 159 161

0 <10 <10 < 10 <10 <10 <10 1 <10 < 10 <10 < 10 <10 <10 2 < 10 < 10 <10 <10 < 10 < 10 3 <10 <10 <10 <10 < 10 <10 4 <10 <10 < 10 < 10 <10 <10 5 <10 <,10 < 10 <10 <10 <10 6 <10 <10 <10 <10 < 10 <10 7 <10 <10 <10 < 10 < 10 <10 8 10 <10 <10 <10 <10 <10 9 10 10 <10 <10 <10 < 10

10 10 10 10 < 10 10 10 11 10 10 10 <10 10 10 12 10 10 10 <10 10 10 13 10 10 10 <10 10 10 14 10 10 10 <10 10 10 15 10 < 10 10 10 10 10 16 10 10 10 <10 10 10 17 <10 <10 10 <10 <10 < 10 18 <10 <10 10 <10 10 10 19 <10 <10 10 10 10 10 20 <10 <10 10 10 10 10 21 <10 <10 10 10 10 <10 24 <10' < 10 10 10 10 10 30 10 <10 10 10 10 10 35 <10 < 10 10 10 100 <10 38 <10 <10 10 10 10 <10 42 <10 <10 10 10 10 <10 45 10 <10 10 10 100 <10

aReciprocal of highest serum dilution that produced at least 90% plaque reduction.

bDay postinoculation.

84

Sentinel goat serological response - SN(TC-83)a

DP lb Goat Number

147 152 157 158 159 161

D <10 < 10 < 10 < 10 <10 < 10 1 <10 <10 <10 <10 <10 < 10 2 < 10 < 10 < 10 <10 <10 <JO 3 <10 < 10 < 10 .. <10 < 10 <10 4 <10 <10 <10 <10 <10 < 10 5 <10 <10 < 10 <10 <10 <10 6 10 <10 < 10 <10 <10 <10 7 100 <10 10 <10 < 10 <10 8 800 10 100 <10 10 10 9 1600 100 400 10 10 100

10 1600 400 400 10 100 200 11 1600 400 400 100 100 800 12 1600 800 1600 400 200 800 13 1600 400 1600 800 400 800 14 800 800 1600 800 400 800 15 800 800 800 800 400 800 16 800 800 1600 1600 800 800 17 800 400 1600 800 400 800 18 400 200 800 800 200 800 19 400 200 400 800 200 800 20 200 200 400 800 400 800 21 400 200 800 1600 200 800 24 800 100 400 800 200 400 30 800 200 400 800 200 800 35 800 100 400 800 4oo 1600 38 400 100 400 800 800 800 42 800 100 200 Boo 800 400 45 400 100 400 800 800 . 800

aReciprocal of highest serum di.lution that produced at least 90% plaque reduction.

bDay postinoculation.

B5

Antiserum production in the goat. Serological response.

Goat Number a Day Postinoculation

Hernrnagglutination-inhibition b

0 21 30 42

149 <10 Boo 1600 Boo 150 <10 -.. <10 4oo 400 155 <10 Boo Boo 200 156 < 10 200 200 100

Serum Neutralization (GJ9-1BJ)c

149 < 10 100 10 <10 150 <10 <10 10 10 155 <10 10 100 <10 156 < 10 10 10 10

Serum Neutralization (TC-B3) c

149 <10 Boo 1600 8oci 150 <10 <10 400 400 155 <10 800 800 200 156 <10 200 200 100

aGroup 1 = 149, 150. Group 2 = 155, 156.

bExpressed as reciprocal of highest serum dilution giving inhibition of hemagglutination.

cReciprocal of highest serum dilution that produced at least 90% plaque reduction.

86

Serological response following hyperimmunization - Hla

DP lb Goat Number 147 149 150 152 155 156

49 640 320 160 < 10 320 40 52 64Ci 20 56 32,000 32,000 59 32,000 32,000 32,000 64,ooo 32,000 32,000 63 32,000 32,000 16,000 66 16,000 32,000 70 4000 64,ooo 16,000 64,ooo 16, 000 16,000 72 64,000 4000 16,000 73 4000 77 4Cioo 80 2000 4000 4000 84 2000 87 2000 90 2000 20 . 2000

DPI Goat Number 157 158 159 160c 161 162

49 320 320 320 < 10 320 <10 52 160 320 320 <10 160 <10 56 32,000 8000 64,ooo 10 64,ooo < 10 59 32,000 8000 64,ooo 640 16,000 <10 63 64,ooo 8000 32,000 1280 16,000 < 10 66 32,000 8000 32,000 2560 16,000 <10 70 32,000 8000 8000 1000 16,000 < 10 72 <10 73 16,.000 4000 1280 <10 77 32,000 2000 5120 <10 80 16,000 2000 2560 <10 84 8000 2000 2560 <10 87 2560 <10 90 8000 2000 2560 <10

aReciprocal of highest serum dilution giving inhibition of hemagglutination.

b Day postinoculation. c . GJ9-1BJ VEE administered 40,000 SMICLD O goat esophagus orig in virus

intravenously\on da9 50.

87

Serological response following hyperimmunization - SN,GJ9-1BJ a

DP lb Goat Number

147 149 150 152 155 156 157 158 159 16oc 161

4g <10 10 100 <10 <10 10 10 10 100 <10 <10 52 <10 80 < 10 <10 100 <10 <10 56 80 1280 320 1280 1280 100 160 59 320 1280 640 1280 160 320 320 320 1280 640 640 63 10 640 100 1280 100 320 100 160 1280 320 160 66 4o 1280 160 80 1280 160 320 70 80 640 320 1280 40 160 20 80 640 100 100 72 100 160 1000 73 20 40 100 100 77 40 80 100 100 80 40 20 100 80 100 20 84 4o 40 40 87 4o 4o 40 90 40 20 80 80 4o 10

aReciprocal of highest serum dilution that produced at least 90% plaque reduction.

bDay postinoculation.

162

< 10 < 10 <10 <10 <10 <10 <10

<10 <10 <10 <10 <10 <10

c4o,ooo SMICLD 0 goat esophagus origin GJ9-1BJ VEE virus administered intravenously on da~ 50.

BB

Serological response following hyperimmunization - SN TC-B3a

DP lb Goat Number

147 149 150 152 155 156

49 400 400 200 100 400 100 52 1600 200 56 51,200 51,200 59 51,200 51,200 12,Boo 25,600 51,200 12,Boo 63 51,200 51,200 12,BOO 12,Boo 51,200· 12,Boo 66 51,200 12,Boo 70 25,600 25,600 6400 12,Boo 12,BOO 12,Boo 72 51,200 12,Boo 12,BOO 73 51,200 77 25,600 Bo 12,BOO 12,Boo 3200 B4 12,BOO B7 12,Boo 90 12,BOO 12,BOO 1600

DPI Goat Number

157 15B 159 16oc 161 162 49 200 4oo Boo 10 400 <10 52 400 Boo Boo 10 Boo <10 56 51,200 51,200 51, 200 Boo 51,200 <10 59 51,200 51,200 51,200 Boo 51,200 <10 63 51,200 51,200 51,200 Boo 51,200 <10 66 51,200 25,600 51,200 200 25,600 <10 70 12,Boo 6400 51,200 200 25,600 <10 72 <10 73 51,200 12,BOO 200 <10 77 25,600 6400 200 <10 Bo 12,BOO 12,Boo 200 <10 B4 12,Boo 6400 200 <10 B7 6400 200 <10 90 12,Boo 6400 200 <10

aReciprocal of highest serum dilution that produced at least 90% plaque reduction. Final titer recorded on day of exanguination with serum harvest.

bDay postinoculatlon.

c4o,ooo SMICLD 0 goat intravenously on da? 50.

esophagus origin GJ9-1BJ VEE virus administered

l.

89

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ACKNOWLEDGEMENTS

The author wishes to thank Doctor c. J. Mare for his counsel and

guidance in the development and completion of this thesis.

This study was conducted with the guidance and general supervision

of Doctor E. A. Carbrey.

The author is indebted to the"'late Doctor E. P.· Pope who through.

his vision established this two-year advanced veterinary· training

program.

The author extends his appreciation to Doctor E. C. Sharman and

the Emergency Programs staff who made this developmental program

possible by their support.

Appreciation is expressed to Doctor J. E. Pearson and Messrs. G.

Gustafson, D. Senne, and M. Snyder for their assistance in the effectuation

of laboratory principles and techniques, and to Mister R. M. Glaz·ier and

his staff for preparation .of the excellent graphs and photographs.